Deprenyl compounds for treatment of glaucoma

Methods and kits for treatment of glaucoma are disclosed. In general, the methods of the invention include administering a therapeutically effective amount of a deprenyl compound to a subject such that the subject is treated for glaucoma.

BACKGROUND OF THE INVENTION 
Glaucoma is a disease of the eye characterized by elevated intraocular 
pressure. The elevated intraocular pressure leads to hardening of the 
eyeball, narrowing of the field of vision and a decrease in a subject's 
visual acuity. Glaucoma is a disease of the optic nerve and the elevated 
eye pressures are related to damage of this nerve. The optic nerve carries 
images from the retina to the brain. Glaucoma damages optical nerve cells 
causing blindspots to occur within a subject's vision. These blind spots 
typically are not noticed by the subject until considerable damage to the 
optic nerve has already occurred. The terminal stage of glaucoma is total 
blindness of the subject. 
Approaches to treating glaucoma include the topical application of 
cholinergic agents, e.g., pilocarpine, alpha- or beta- adrenergic agonists 
or antagonists, e.g., clonidine, timolol or epinephrine. An alternative 
approach for treating glaucoma is the systemic administration of carbonic 
anhydrase inhibitors. In some cases laser or operative surgery is used to 
treat glaucoma. 
Problems exist with the aforementioned approaches to treating glaucoma in 
that the treatments can be accompanied by side-effects. For example the 
instillation of a cholinergic agent, such as pilocarpine, into the eye of 
a subject can cause nausea, diarrhea, muscular spasms, sweating, 
lacrimation, salivation, etc. Contraction of the pupil (myosis) and of the 
ciliary muscle of the eye, as well as dilation of the blood vessels of the 
iris and conjunctiva also can be observed. Visual complications, e.g., 
spasm of accommodation, myopia or a decrease in visual acuity, also can 
occur. 
The treatment with a sympathomimetic agent such as dipivalylepinephrine is 
known frequently to produce sensations of burning or irritation in a 
subject. Another side-effect of these agents is the appearance of cardiac 
disturbances, e.g., palpitations, tachycardia, arrythmia, etc. 
Clonidine, which is known as an alpha-2-adrenergic receptor agonist, can 
bring about mydriasis, as well as an initial phase of ocular hypertension 
(biphasic effect). Furthermore, in spite of the topical application of the 
product to the eye, important systemic effects, such as bradycardia and 
hypotension, have been observed. 
The use of beta-blocking medicaments also can cause important systemic 
effects after topical administration to the eye, due to the absence of a 
"first pass effect". Timolol, for example, causes bradycardia or 
hypotension. These systemic secondary reactions to beta-blocking 
medicaments can reach such a severe level that the treatment has to be 
discontinued. Cases of suicidal depression, hallucinations, nightmares or 
psychoses requiring hospitalization have been reported in connection with 
these medicaments. Furthermore, these compounds have to be administered 
with extreme precautions to patients subject to cardiac or pulmonary 
functional disorders. In such patients, amongst others, cases of 
arrhythmia, cardiac arrest, asthma, dyspnea and bronchospasms have been 
reported. 
The treatment with a sympatholytic agent, such as guanethidine, causes 
hyperemia of the conjunctiva and some irritation, not to mention the fact 
that these agents only have a low tendency to reduce intraocular pressure. 
Finally, in the treatment of glaucoma with carbonic anhydrase inhibitors, 
such as acetazolamide or methazolamide, serious systemic side-effects, 
such as depression of the central nervous system, weight loss and, mainly, 
bone marrow hypofunction, have been reported. 
The use of conventional hypotensive agents for the treatment of glaucoma is 
accompanied by considerable risks. Known medications are not particularly 
well suited for topical treatment and the systemic side-effects of these 
medicaments make them delicate to use because these effects are far from 
being negligible and because they can have, in some cases, severe 
consequences. 
SUMMARY OF THE INVENTION 
This invention provides methods and kits useful for the treatment of 
glaucoma. In one aspect, the methods of the invention include 
administering a therapeutically effective amount of a deprenyl compound to 
a subject such that the subject is treated for glaucoma. In a preferred 
embodiment, the deprenyl compound is represented by the structure: 
##STR1## 
in which R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, 
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R.sub.2 
is hydrogen or alkyl; R.sub.3 is a single bond, alkylene, or 
--(CH.sub.2).sub.n --X--(CH.sub.2).sub.m in which X is O, S, or N-methyl; 
m is 1 or 2, and n is 0,1, or 2; R.sub.4 is alkyl, alkenyl, alkynyl, 
heterocyclyl, aryl or aralkyl; R.sub.5 is alkylene, alkenylene, alkynylene 
and alkoxylene; and R.sub.6 is C.sub.3 -C.sub.6 cycloalkyl or 
EQU .sup.--C.tbd.CH ; or 
R.sub.2 and R.sub.4 -R.sub.3 are joined to form, together with the methine 
to which they are attached, a cyclic or polycyclic group; and 
pharmaceutically acceptable salts thereof. In preferred embodiments, 
R.sub.1 is a group that can be removed in vivo; R.sub.1 is hydrogen; 
R.sub.1 is alkyl; R.sub.1 is methyl; R.sub.2 is methyl; R.sub.3 is 
methylene; R.sub.4 is aryl; or R.sub.4 is phenyl. In still other preferred 
embodiments, R.sub.5 is alkylene, more preferably methylene. In other 
preferred embodiments, R.sub.6 is .sup.--C.tbd.CH . In other preferred 
embodiments, R.sub.6 is cyclopentyl. 
In another embodiment, the deprenyl compound has the structure 
##STR2## 
in which R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, 
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl. 
In another preferred embodiment, the deprenyl compound is represented by 
the structure: 
##STR3## 
in which R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, 
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R.sub.2 
is hydrogen or alkyl; R.sub.3 is a bond or methylene; and R.sub.4 is aryl 
or aralkyl; or R.sub.2 and R.sub.4 -R.sub.3 are joined to form, together 
with the methine to which they are attached, a cyclic or polycyclic group; 
and pharmaceutically acceptable salts thereof. 
In another embodiment, the deprenyl compound is represented by the 
structure: 
##STR4## 
in which 
R.sub.2 is hydrogen or alkyl; R.sub.3 is a bond or methylene; and R.sub.4 
is aryl or aralkyl; or R.sub.2 and R.sub.4 -R.sub.3 are joined to form, 
together with the methine to which they are attached, a cyclic or 
polycyclic group; and R.sub.5 is alkylene, alkenylene, alkynylene and 
alkoxylene; and pharmaceutically acceptable salts thereof. 
In yet another embodiment, the deprenyl compound is represented by the 
structure: 
##STR5## 
in which R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, 
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; A is a 
substituent independently selected for each occurence from the group 
consisting of halogen, hydroxyl, alkyl, alkoxyl, cyano, nitro, amino, 
carboxyl, --CF.sub.3, or azido; n is 0 or an integer from 1 to 5; and 
pharmaceutically acceptable salts thereof. 
In other preferred embodiments, the deprenyl compound is (-)-deprenyl, 
(-)-pargyline, or (-)-desmethyldeprenyl. 
In another aspect, tie invention provides a kit useful for the treatment of 
glaucoma. In one embodiment, the kit includes a container of a deprenyl 
compound and instructions for administering a therapeutically effective 
amount of the deprenyl compound to a subject such that the subject is 
treated for glaucoma.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides methods of treating glaucoma. In general, 
the methods include administering a therapeutically effective amount of a 
deprenyl compound to a subject in need thereof, such that the subject is 
treated for glaucoma. 
The language "glaucoma" is art-recognized. The term includes both acute and 
chronic diseases of the eye characterized by elevated intraocular 
pressure. Symptoms of glaucoma include high pressure within the eyeball, 
hardening of the eyeball, narrowing of the field of vision, death of optic 
nerve cells, development of retinal blind spots, and decreased visual 
acuity. 
The term "subject", as used herein, refers to a warm-blooded animal in need 
of treatment for, or susceptible to, glaucoma. In preferred embodiments, 
the subject is a mammal, including humans and non-human mammals such as 
dogs, cats, pigs, cows, sheep, goats, rats, and mice. In a particularly 
preferred embodiment, the subject is a human. 
The language "therapeutically effective amount" of a deprenyl compound, as 
used herein, refers to an amount of a therapeutic compound sufficient to 
significantly ameliorate glaucoma or at least one symptom thereof in a 
subject. "Significant amelioration" includes elimination or substantial 
reduction in severity of one or more symptoms or diagnostic 
characteristics of glaucoma. A "substantial reduction" means at least 
about 5% reduction, more preferably at least about 10% reduction, and more 
preferably at least about 20% reduction in severity of one or more 
symptoms or diagnostic characteristics of glaucoma. Thus, a 
therapeutically effective amount of a therapeutic compound can decrease 
intraocular pressure, prevent or delay death of retinal or optic nerve 
cells, improve field of vision or visual acuity, or otherwise ameliorate 
glaucoma in a subject. One of ordinary skill in the art would be able to 
determine such amounts based on such factors as the subject's size, the 
severity of the subject's symptoms, and the particular deprenyl compound 
or route of administration selected. 
I. Deprenyl Compounds 
The language "deprenyl compound", as used herein, includes deprenyl 
(N,.alpha.-dimethyl-N -2-propynylphenethylamine), compounds which are 
structurally similar to deprenyl, e.g., structural analogs, or derivatives 
thereof. Thus, in one embodiment, a deprenyl compound can be represented 
by the following formula (Formula I ): 
##STR6## 
in which 
R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl, 
arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; 
R.sub.2 is hydrogen or alkyl; 
R.sub.3 is a single bond, alkylene, or --(CH.sub.2).sub.n 
--X--(CH.sub.2).sub.m ; 
in which X is O, S, or N-methyl; m is 1 or 2; and n is 0,1, or 2; 
R.sub.4 is alkyl, alkenyl, alkynyl, heterocyclyl, aryl or aralkyl; and 
R.sub.5 is alkylene, alkenylene, alkynylene and alkoxylene; and 
R.sub.6 is C.sub.3 -C.sub.6 cycloalkyl or 
EQU .sup.--C.tbd.CH ; or 
R.sub.2 and R.sub.4 - R.sub.3 are joined to form, together with the methine 
to which they are attached, a cyclic or polycyclic group; 
and pharmaceutically acceptable salts thereof. 
In preferred embodiments, R.sub.1 is a group that can be removed in vivo. 
In certain embodiments, R.sub.1 is hydrogen. In other preferred 
embodiments, R.sub.1 is methyl. In certain preferred embodiments, R.sub.2 
is hydrogen. In certain preferred embodiments, R.sub.2 is methyl. In some 
preferred embodiments, R.sub.3 is alkylene, more preferably methylene. In 
other preferred embodiments, R.sub.3 is --(CH.sub.2).sub.n 
--X--(CH.sub.2).sub.m. In preferred embodiments, R.sub.4 is aryl. In 
certain preferred embodiments, R.sub.4 is phenyl. In other preferred 
embodiments, R.sub.4 is aralkyl. In yet other preferred embodiments, 
R.sub.4 is alkyl. In still other preferred embodiments, R.sub.5 is 
alkylene, more preferably methylene. In certain preferred embodiments, 
R.sub.6 is 
EQU --C.tbd.CH 
In other preferred embodiments, R.sub.6 is cyclopentyl. 
In another preferred embodiment, the deprenyl compound has the structure 
##STR7## 
wherein R.sub.1 is as described above. In other preferred embodiments, the 
deprenyl compound is AGN-1133 (N-methyl-N-propynyl-1-indanamine) or 
AGN-1135 (N-propynyl-1-indanamine). Preferred deprenyl compounds include 
(-)-deprenyl, (-)-pargyline, (-)-desmethyldeprenyl, and 
##STR8## 
In another embodiment, a deprenyl compound can be represented by the 
following formula (Formula II): 
##STR9## 
in which 
R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl, 
arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; 
R.sub.2 is hydrogen or alkyl; 
R.sub.3 is a bond or methylene; and 
R.sub.4 is aryl or aralkyl; or 
R.sub.2 and R.sub.4 -R.sub.3 are joined to form, together with the methine 
to which they are attached, a cyclic or polycyclic group; 
and pharmaceutically acceptable salts thereof. 
In another embodiment, the deprenyl compound can be represented by the 
following formula (Formula III): 
##STR10## 
in which 
R.sub.2 is hydrogen or alkyl; 
R.sub.3 is a bond or methylene; and 
R.sub.4 is aryl or aralkyl; or 
R.sub.2 and R.sub.4 -R.sub.3 are joined to form, together with the methine 
to which they are attached, a cyclic or polycyclic group; and 
R.sub.5 is alkylene, alkenylene, alkynylene and alkoxylene; 
and pharmaceutically acceptable salts thereof. 
In yet another embodiment, the deprenyl compound can be represented by the 
following formula (Formula IV): 
##STR11## 
in which 
R.sub.1 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl, 
arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; 
A is a substituent independently selected for each occurence from the group 
consisting of halogen, hydroxyl, alkyl, alkoxyl, cyano, nitro, amino, 
carboxyl, --CF.sub.3, or azido; 
n is 0 or an integer from 1 to 5; 
and pharmaceutically acceptable salts thereof. 
In certain embodiments of the invention, the deprenyl compound is not 
deprenyl (including (-)-deprenyl). 
The term "alkyl" refers to the radical of saturated aliphatic groups, 
including straight chain alkyl groups, branched-chain alkyl groups, 
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and 
cycloalkyl substituted alkyl groups. In preferred embodiments, a straight 
chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone 
(e.g., C.sub.1 -C.sub.20 for straight chain, C.sub.3 -C.sub.20 for 
branched chain), and more preferably 10 or fewer. Likewise, preferred 
cycloalkyls have from 4-10 carbon atoms in their ring structure, and more 
preferably have 5, 6 or 7 carbons in the ring structure. Unless the number 
of carbons is otherwise specified, "lower alkyl" as used herein means an 
alkyl group, as defined above, but having from one to six carbon atoms in 
its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have 
similar chain lengths. Preferred alkyl groups are lower alkyls. In 
preferred embodiments, a substituent designated herein as alkyl is a lower 
alkyl. 
Moreover, the term "alkyl" (or "lower alkyl") as used throughout the 
specification and claims is intended to include both "unsubstituted 
alkyls" and "substituted alkyls", the latter of which refers to alkyl 
moieties having substituents replacing a hydrogen on one or more carbons 
of the hydrocarbon backbone. Such substituents can include, for example, 
halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including 
alkylcarbonyl and arylcarbonyl groups), and esters (including 
alkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl, acyloxy, 
alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, 
amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, 
sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic 
or heteroaromatic moiety. It will be understood by those skilled in the 
art that the moieties substituted on the hydrocarbon chain can themselves 
be substituted, if appropriate. For instance, the substituents of a 
substituted alkyl may include substituted and unsubstituted forms of 
aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and 
phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and 
sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls 
(including ketones, aldehydes, carboxylates, and esters), --CF.sub.3, --CN 
and the like. Exemplary substituted alkyls are described below. 
Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, 
alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF.sub.3, --CN, 
and the like. 
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups 
analogous in length and possible substitution to the alkyls described 
above, but that contain at least one double or triple bond respectively. 
The term "aralkyl", as used herein, refers to an alkyl or alkylenyl group 
substituted with at least one aryl group (e.g., an aromatic or 
heteroaromatic group). Exemplary aralkyls include benzyl (i.e., 
phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and 
the like. 
The term "alkylcarbonyl", as used herein, refers to --C(O)-alkyl. 
Similarly, the term "arylcarbonyl" refers to --C(O)--aryl. The term 
"alkyloxycarbonyl", as used herein, refers to the group --C(O)--O--alkyl, 
and the term "aryloxycarbonyl" refers to --C(O)--O--aryl. The term 
"acyloxy" refers to --O--C(O)--R.sub.7, in which R.sub.7 is alkyl, 
alkenyl, alkynyl, aryl, aralkyl or heterocyclyl. 
The term "amino", as used herein, refers to --N(R.sub.8)(R.sub.9), in which 
R.sub.8 and R.sub.9 are each independently hydrogen, alkyl, alkyenyl, 
alkynyl, aralkyl, aryl, or R.sub.8 and R.sub.9, together with the nitrogen 
atom to which they are attached, form a ring having 4-8 atoms. Thus, the 
term "amino", as used herein, includes unsubstituted, monosubstituted 
(e.g., monoalkylamino or monoarylamino), and disubstituted (e.g., 
dialkylamino or alkylarylamino) amino groups. The term "amido" refers to 
--C(O)--N(R.sub.8)(R.sub.9), in which R.sub.8 and R.sub.9 are as defined 
above. The term "acylamino" refers to --N(R'.sub.8)C(O)--R.sub.7, in which 
R.sub.7 is as defined above and R'.sub.8 is alkyl. 
As used herein, the term "nitro" means --NO.sub.2 ; the term "halogen" 
designates --F, --Cl, --Br or --I; the term "sulfhydryl" means --SH; and 
the term "hydroxyl" means --OH. 
The term "aryl" as used herein includes 5-, 6- and 7-membered aromatic 
groups that may include from zero to four heteroatoms in the ring, for 
example, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, 
thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and 
pyrimidinyl, and the like. Those aryl groups having heteroatoms in the 
ring structure may also be referred to as "aryl heterocycles" or 
"heteroaromatics". The aromatic ring can be substituted at one or more 
ring positions with such substituents as described above, as for example, 
halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, 
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, 
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, 
ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic 
moiety, --CF.sub.3, --CN, or the like. Aryl groups can also be part of a 
polycyclic group. For example, aryl groups include fused aromatic moieties 
such as naphthyl, anthracenyl, quinolyl, indolyl, and the like. 
The terms "heterocyclyl" or "heterocyclic group" refer to 4- to 10-membered 
ring structures, more preferably 4- to 7-membered rings, which ring 
structures include one to four heteroatoms. Heterocyclyl groups include, 
for example, pyrrolidine, oxolane, thiolane, imidazole, oxazole, 
piperidine, piperazine, morpholine, lactones, lactams such as azetidinones 
and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring 
can be substituted at one or more positions with such substituents as 
described above, as for example, halogen, alkyl, aralkyl, alkenyl, 
alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, 
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, 
sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or 
heteroaromatic moiety, --CF.sub.3, --CN, or the like. 
The terms "polycyclyl" or "polycyclic group" refer to two or more rings 
(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or 
heterocyclyls) in which two or more carbons are common to two adjoining 
rings, e.g., the rings are "fused rings". Rings that are joined through 
non-adjacent atoms are termed "bridged" rings. Each of the rings of the 
polycyclic group can be substituted with such substituents as described 
above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, 
cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, 
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, 
ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic 
moiety, --CF.sub.3, --CN, or the like. 
The term "heteroatom" as used herein means an atom of any element other 
than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, 
sulfur and phosphorus. 
It will be noted that the structure of some of the compounds of this 
invention includes asymmetric carbon atoms. It is to be understood 
accordingly that the isomers arising from such asymmetry are included 
within the scope of this invention. Such isomers are obtained in 
substantially pure form by classical separation techniques and by 
sterically controlled synthesis 
The term "can be removed in vivo", as used herein, refers to a group that 
can be cleaved in vivo, either enzymatically or non-enzymatically. For 
example, amides can be cleaved by amidases, and N-methyl amines can be 
cleaved by enzymatic oxidation. For example, when deprenyl is administered 
to a subject, it is believed, as described infra, that the methyl group 
can be removed in vivo to yield an active compound. As a further example, 
with reference to Formula I, when R.sub.1 is alkylcarbonyl, the resulting 
amide group can be cleaved in vivo to yield a secondary amine (e.g., 
R.sub.1 is converted to hydrogen in vivo). Other groups which can be 
removed in vivo are known (see, e.g., R. B. Silverman (1992) "The Organic 
Chemistry of Drug Design and Drug Action", Academic Press, San Diego) and 
can be employed in compounds useful in the present invention. 
II. Pharmaceutical Compositions 
The phrase "pharmaceutically acceptable" is employed herein to refer to 
those compounds, materials, compositions, and/or dosage forms which are, 
within the scope of sound medical judgment, suitable for use in contact 
with the tissues of human beings and animals without excessive toxicity, 
irritation, allergic response, or other problem or complication, 
commensurate with a reasonable benefit/risk ratio. 
The phrase "pharmaceutically-acceptable carrier" as used herein means a 
pharmaceutically-acceptable material, composition or vehicle, such as a 
liquid or solid filler, diluent, excipient, solvent or encapsulating 
material, involved in carrying or transporting the subject deprenyl 
compound from one organ, or portion of the body, to another organ, or 
portion of the body. Each carrier must be "acceptable" in the sense of 
being compatible with the other ingredients of the formulation and not 
injurious to the patient. Some examples of materials which can serve as 
pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, 
glucose and sucrose; (2) starches, such as corn starch and potato starch; 
(3) cellulose, and its derivatives, such as sodium carboxymethyl 
cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; 
(5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and 
suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower 
oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such 
as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol 
and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl 
laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and 
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) 
isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) 
phosphate buffer solutions; and (21) other non-toxic compatible substances 
employed in pharmaceutical formulations. 
The stability of deprenyl can be affected by the pH of the medium in which 
the deprenyl is formulated. For example, deprenyl is more stable at a pH 
in the range of about 3-5 than at a pH of about 7. Therefore, when 
formulating a deprenyl compound in a pharmaceutical composition, it is 
preferred that the deprenyl compound be maintained at a suitable pH. In 
preferred embodiments, a pharmaceutical composition of the invention has a 
pH in the range of about 3 to about 5, more preferably about 3 to about 4. 
Furthermore, ethyl alcohol is a preferred solvent for improving stability 
of deprenyl. Thus, in certain embodiments, alcoholic or aqueous alcoholic 
media are preferred for the pharmaceutical compositions of the invention. 
As set out above, certain embodiments of the present deprenyl compounds may 
contain a basic functional group, such as amino or alkylamino, and are, 
thus, capable of forming pharmaceutically-acceptable salts with 
pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable 
salts" in this respect, refers to the relatively non-toxic, inorganic and 
organic acid addition salts of compounds of the present invention. These 
salts can be prepared in situ during the final isolation and purification 
of the compounds of the invention, or by separately reacting a purified 
compound of the invention in its free base form with a suitable organic or 
inorganic acid, and isolating the salt thus formed. Representative salts 
include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, 
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, 
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, 
succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, 
and laurylsulfonate salts and the like (see, for example, Berge et al. 
(1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19). 
In other cases, the deprenyl compounds of the present invention may contain 
one or more acidic functional groups and, thus, are capable of forming 
pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. 
The term "pharmaceutically-acceptable salts" in these instances refers to 
the relatively non-toxic, inorganic and organic base addition salts of 
compounds of the present invention. These salts can likewise be prepared 
in situ during the final isolation and purification of the compounds, or 
by separately reacting the purified compound in its free acid form with a 
suitable base, such as the hydroxide, carbonate or bicarbonate of a 
pharmaceutically-acceptable metal cation, with ammonia, or with a 
pharmaceutically-acceptable organic primary, secondary or tertiary amine. 
Representative alkali or alkaline earth salts include the lithium, sodium, 
potassium, calcium, magnesium, and aluminum salts and the like. 
Representative organic amines useful for the formation of base addition 
salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, 
diethanolamine, piperazine and the like (see, for example, Berge et al., 
supra). 
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate 
and magnesium stearate, as well as coloring agents, release agents, 
coating agents, sweetening, flavoring and perfuming agents, preservatives 
and antioxidants can also be present in the compositions. 
Examples of pharnaceutically-acceptable antioxidants include: (1) water 
soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, 
sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) 
oil-soluble antioxidants, such as ascorbyl palmitate, butylated 
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl 
gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, 
such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, 
tartaric acid, phosphoric acid, and the like. 
Formulations of the present invention include those suitable for oral, 
nasal, topical (including buccal and sublingual), rectal, vaginal and/or 
parenteral administration. The formulations may conveniently be presented 
in unit dosage form and may be prepared by any methods well known in the 
art of pharmacy. The amount of active ingredient which can be combined 
with a carrier material to produce a single dosage form will vary 
depending upon the host being treated, the particular mode of 
administration. The amount of active ingredient which can be combined with 
a carrier material to produce a single dosage form will generally be that 
amount of the deprenyl compound which produces a therapeutic effect. 
Generally, out of one hundred per cent, this amount will range from about 
0.01 per cent to about ninety-nine percent of active ingredient, 
preferably from about 0. 1 per cent to about 70 per cent, most preferably 
from about 1 per cent to about 30 per cent. 
Methods of preparing these formulations or compositions include the step of 
bringing into association a deprenyl compound of the present invention 
with the carrier and, optionally, one or more accessory ingredients. In 
general, the formulations are prepared by uniformly and intimately 
bringing into association a deprenyl compound of the present invention 
with liquid carriers, or finely divided solid carriers, or both, and then, 
if necessary, shaping the product. 
Formulations of the invention suitable for oral administration may be in 
the form of capsules, cachets, pills, tablets, lozenges (using a flavored 
basis, usually sucrose and acacia or tragacanth), powders, granules, or as 
a solution or a suspension in an aqueous or non-aqueous liquid, or as an 
oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or 
as pastilles (using an inert base, such as gelatin and glycerin, or 
sucrose and acacia) and/or as mouth washes and the like, each containing a 
predetermined amount of a compound of the present invention as an active 
ingredient. A deprenyl compound of the present invention may also be 
administered as a bolus, electuary or paste. 
In solid dosage forms of the invention for oral administration (capsules, 
tablets, pills, dragees, powders, granules and the like), the active 
ingredient is mixed with one or more pharmaceutically-acceptable carriers, 
such as sodium citrate or dicalcium phosphate, and/or any of the 
following: (1) fillers or extenders, such as starches, lactose, sucrose, 
glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, 
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose 
and/or acacia; (3) humectants, such as glycerol; (4) disintegrating 
agents, such as agar-agar, calcium carbonate, potato or tapioca starch, 
alginic acid, certain silicates, and sodium carbonate; (5) solution 
retarding agents, such as paraffin; (6) absorption accelerators, such as 
quaternary ammonium compounds; (7) wetting agents, such as, for example, 
cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin 
and bentonite clay; (9) lubricants, such a talc, calcium stearate, 
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and 
mixtures thereof; and (10) coloring agents. In the case of capsules, 
tablets and pills, the pharmaceutical compositions may also comprise 
buffering agents. Solid compositions of a similar type may also be 
employed as fillers in soft and hard-filled gelatin capsules using such 
excipients as lactose or milk sugars, as well as high molecular weight 
polyethylene glycols and the like. 
A tablet may be made by compression or molding, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared using binder 
(for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert 
diluent, preservative, disintegrant (for example, sodium starch glycolate 
or cross-linked sodium carboxymethyl cellulose), surface-active or 
dispersing agent. Molded tablets may be made by molding in a suitable 
machine a mixture of the powdered deprenyl compound moistened with an 
inert liquid diluent. 
The tablets, and other solid dosage forms of the pharmaceutical 
compositions of the present invention, such as dragees, capsules, pills 
and granules, may optionally be scored or prepared with coatings and 
shells, such as enteric coatings and other coatings well known in the 
pharmaceutical-formulating art. They may also be formulated so as to 
provide slow or controlled release of the active ingredient therein using, 
for example, hydroxypropylmethyl cellulose in varying proportions to 
provide the desired release profile, other polymer matrices, liposomes 
and/or microspheres. They may be sterilized by, for example, filtration 
through a bacteria-retaining filter, or by incorporating sterilizing 
agents in the form of sterile solid compositions which can be dissolved in 
sterile water, or some other sterile injectable medium immediately before 
use. These compositions may also optionally contain opacifying agents and 
may be of a composition that they release the active ingredient(s) only, 
or preferentially, in a certain portion of the gastrointestinal tract, 
optionally, in a delayed manner. Examples of embedding compositions which 
can be used include polymeric substances and waxes. The active ingredient 
can also be in micro-encapsulated form, if appropriate, with one or more 
of the above-described excipients. 
Liquid dosage forms for oral administration of the deprenyl compounds of 
the invention include pharmaceutically acceptable emulsions, 
microemulsions, solutions, suspensions, syrups and elixirs. In addition to 
the active ingredient, the liquid dosage forms may contain inert diluents 
commonly used in the art, such as, for example, water or other solvents, 
solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl 
alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 
propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, 
groundnut, corn, germ, olive, castor and sesame oils), glycerol, 
tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of 
sorbitan, and mixtures thereof. 
Besides inert diluents, the oral compositions can also include adjuvants 
such as wetting agents, emulsifying and suspending agents, sweetening, 
flavoring, coloring, perfuming and preservative agents. 
Suspensions, in addition to the active deprenyl compound, may contain 
suspending agents as, for example, ethoxylated isostearyl alcohols, 
polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, 
aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures 
thereof. 
Formulations of the pharmaceutical compositions of the invention for rectal 
or vaginal administration may be presented as a suppository, which may be 
prepared by mixing one or more deprenyl compounds of the invention with 
one or more suitable nonirritating excipients or carriers comprising, for 
example, cocoa butter, polyethylene glycol, a suppository wax or a 
salicylate, and which is solid at room temperature, but liquid at body 
temperature and, therefore, will melt in the rectum or vaginal cavity and 
release the deprenyl compound. 
Formulations of the present invention which are suitable for vaginal 
administration also include pessaries, tampons, creams, gels, pastes, 
foams or spray formulations containing such carriers as are known in the 
art to be appropriate. 
Dosage forms for the topical or transdermal administration of a deprenyl 
compound of this invention include powders, sprays, ointments, pastes, 
creams, lotions, gels, solutions, patches and inhalants. The active 
compound may be mixed under sterile conditions with a 
pharmaceutically-acceptable carrier, and with any preservatives, buffers, 
or propellants which may be required. 
The ointments, pastes, creams and gels may contain, in addition to a 
deprenyl compound of this invention, excipients, such as animal and 
vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose 
derivatives, polyethylene glycols, silicones, bentonites, silicic acid, 
talc and zinc oxide, or mixtures thereof. 
Powders and sprays can contain, in addition to a compound of this 
invention, excipients such as lactose, talc, silicic acid, aluminum 
hydroxide, calcium silicates and polyamide powder, or mixtures of these 
substances. Sprays can additionally contain customary propellants, such as 
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as 
butane and propane. 
Transdermal patches have the added advantage of providing controlled 
delivery of a compound of the present invention to the body. Such dosage 
forms can be made by dissolving or dispersing the deprenyl compound in the 
proper medium. Absorption enhancers can also be used to increase the flux 
of the deprenyl compound across the skin. The rate of such flux can be 
controlled by either providing a rate controlling membrane or dispersing 
the deprenyl compound in a polymer matrix or gel. Devices, including 
patches, which transdermally deliver a deprenyl compound by iontophoresis 
or other electrically-assisted methods can also be employed in the present 
invention, including, for example, the devices described in U.S. Pat. Nos. 
4,708,716 and 5,372,579. 
Ophthalmic formulations, eye ointments, powders, solutions, drops, sprays 
and the like, are also contemplated as being within the scope of this 
invention. 
Pharmaceutical compositions of this invention suitable for parenteral 
administration comprise one or more deprenyl compounds of the invention in 
combination with one or more pharmaceutically-acceptable sterile isotonic 
aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or 
sterile powders which may be reconstituted into sterile injectable 
solutions or dispersions just prior to use, which may contain 
antioxidants, buffers, bacteriostats, solutes which render the formulation 
isotonic with the blood of the intended recipient or suspending or 
thickening agents. 
Examples of suitable aqueous and nonaqueous carriers which may be employed 
in the pharmaceutical compositions of the invention include water, 
ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, 
and the like), and suitable mixtures thereof, vegetable oils, such as 
olive oil, and injectable organic esters, such as ethyl oleate. Proper 
fluidity can be maintained, for example, by the use of coating materials, 
such as lecithin, by the maintenance of the required particle size in the 
case of dispersions, and by the use of surfactants. 
These compositions may also contain adjuvants such as preservatives, 
wetting agents, emulsifying agents and dispersing agents. Prevention of 
the action of microorganisms may be ensured by the inclusion of various 
antibacterial and antifungal agents, for example, paraben, chlorobutanol, 
phenol sorbic acid, and the like. It may also be desirable to include 
isotonic agents, such as sugars, sodium chloride, and the like into the 
compositions. In addition, prolonged absorption of the injectable 
pharmaceutical form may be brought about by the inclusion of agents which 
delay absorption such as aluminum monostearate and gelatin. 
In some cases, in order to prolong the effect of a drug, it is desirable to 
slow the absorption of the drug from subcutaneous or intramuscular 
injection. This may be accomplished by the use of a liquid suspension of 
crystalline or amorphous material having poor water solubility. The rate 
of absorption of the drug then depends upon its rate of dissolution which, 
in turn, may depend upon crystal size and crystalline form. Alternatively, 
delayed absorption of a parenterally-administered drug form is 
accomplished by dissolving or suspending the drug in an oil vehicle. 
Injectable depot forms are made by forming microencapsule matrices of the 
subject deprenyl compounds in biodegradable polymers such as 
polylactide-polyglycolide. Depending on the ratio of drug to polymer, and 
the nature of the particular polymer employed, the rate of drug release 
can be controlled. Examples of other biodegradable polymers include 
poly(orthoesters) and poly(anhydrides). Depot injectable formulations are 
also prepared by entrapping the drug in liposomes or microemulsions which 
are compatible with body tissue. 
When the compounds of the present invention are administered as 
pharmaceuticals, to humans and animals, they can be given alone or as a 
pharmaceutical composition containing, for example, 0.01 to 99.5% (more 
preferably, 0.1 to 90%) of active ingredient in combination with a 
pharmaceutically acceptable carrier. 
The preparations of the present invention may be given orally, 
parenterally, topically, or rectally. They are of course given by forms 
suitable for each administration route. For example, they are administered 
in tablets or capsule form, by injection, inhalation, eye lotion, 
ointment, suppository, etc.; administration by injection, infusion or 
inhalation; topical by lotion or ointment; and rectal by suppositories. 
Injection (subcutaneous or intraperitoneal) or topical ophthalmic 
administration are preferred. 
The phrases "parenteral administration" and "administered parenterally" as 
used herein means modes of administration other than enteral and topical 
administration, usually by injection, and includes, without limitation, 
intravenous, intramuscular, intraarterial, intrathecal, intracapsular, 
intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, 
subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, 
intraspinal and intrasternal injection and infusion. 
The phrases "systemic administration," "administered systemically," 
"peripheral administration" and "administered peripherally" as used herein 
mean the administration of a compound, drug or other material other than 
directly into the central nervous system, such that it enters the 
patient's system and, thus, is subject to metabolism and other like 
processes, for example, subcutaneous administration. 
These compounds may be administered to humans and other animals for therapy 
by any suitable route of administration, including orally, nasally, as by, 
for example, a spray, rectally, intravaginally, parenterally, 
intracisternally and topically, as by powders, ointments or drops, 
including buccally and sublingually. 
Regardless of the route of administration selected, the compounds of the 
present invention, which may be used in a suitable hydrated form, and/or 
the pharmaceutical compositions of the present invention, are formulated 
into pharnaceutically-acceptable dosage forms by conventional methods 
known to those of skill in the art. 
Actual dosage levels of the active ingredients in the pharmaceutical 
compositions of this invention may be varied so as to obtain an amount of 
the active ingredient which is effective to achieve the desired 
therapeutic response for a particular patient, composition, and mode of 
administration, without being toxic to the patient. 
The selected dosage level will depend upon a variety of factors including 
the activity of the particular deprenyl compound of the present invention 
employed, or the ester, salt or amide thereof, the route of 
administration, the time of administration, the rate of excretion of the 
particular compound being employed, the duration of the treatment, other 
drugs, compounds and/or materials used in combination with the particular 
deprenyl compound employed, the age, sex, weight, condition, general 
health and prior medical history of the patient being treated, and like 
factors well known in the medical arts. 
A physician or veterinarian having ordinary skill in the art can readily 
determine and prescribe the effective amount of the pharmaceutical 
composition required. For example, the physician or veterinarian could 
start doses of the compounds of the invention employed in the 
pharmaceutical composition at levels lower than that required in order to 
achieve the desired therapeutic effect and gradually increase the dosage 
until the desired effect is achieved. 
In general, a suitable daily dose of a deprenyl compound of the invention 
will be that amount of the compound which is the lowest dose effective to 
produce a therapeutic effect. Such an effective dose will generally depend 
upon the factors described above. Generally, intraperitoneal and 
subcutaneous doses of the compounds of this invention for a patient, when 
used for the indicated anti-glaucoma effects, will range from about 0.0001 
to about 10 mg per kilogram of body weight per day, more preferably from 
about 0.001 mg/kg to about 1 mg/kg per day. 
If desired, the effective daily dose of a deprenyl compound may be 
administered as two, three, four, five, six or more sub-doses administered 
separately at appropriate intervals throughout the day, optionally, in 
unit dosage forms. 
While it is possible for a compound of the present invention to be 
administered alone, it is preferable to administer the compound as a 
pharmaceutical formulation (composition). 
Therapeutic compositions can be administered with medical devices known in 
the art. For example, in a preferred embodiment, a therapeutic composition 
of the invention can be administered with a needleless hypodermic 
injection device, such as the devices disclosed in U.S. Pat. Nos. 
5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 
4,596,556. Examples of well-known implants and modules useful in the 
present invention include: U.S. Pat. No. 4,487,603, which discloses an 
implantable micro-infusion pump for dispensing medication at a controlled 
rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for 
administering medicants through the skin; U.S. Pat. No. 4,447,233, which 
discloses a medication infusion pump for delivering medication at a 
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable 
flow implantable infusion apparatus for continuous drug delivery; U.S. 
Pat. No. 4,439,196, which discloses an osmotic drug delivery system having 
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses 
an osmotic drug delivery system. These patents are incorporated herein by 
reference. Many other such implants, delivery systems, and modules are 
well known to those skilled in the art. 
It is believed that certain deprenyl compounds can be at least partially 
metabolized in vivo after administration to a subject. For example, 
(-)-deprenyl can be metabolized by the liver to (-)-desmethyldeprenyl, as 
well as (-)-methamphetamine and (-)-amphetamine, after oral 
administration. The hepatic metabolism of (-)-deprenyl can be inhibited by 
administration of a P.sub.450 inhibitor such as Proadifen. In animal and 
cell-culture studies, administration of Proadifen reduces the ability of 
(-)-deprenyl to prevent cell death, but does not block the cell-rescuing 
activity of (-)-desmethyldeprenyl. Thus, it is believed that at least one 
metabolite of (-)-deprenyl, most likely (-)-desmethyldeprenyl, is an 
active compound. It is presently believed that (-)-methamphetamine and 
(-)-amphetamine are inhibitors of the cell-rescuing activity of deprenyl 
compounds. It is also believed that monoamine oxidase (MAO, including both 
MAO-A and MAO-B) inhibitory activity is not required for treatment of 
glaucoma. Absence of MAO inhibitor activity may in fact provide a drug 
with fewer side effects. Thus, in certain embodiments, it is preferred 
that the deprenyl compound have low MAO inhibitor activity, or be 
administered so as to minimize MAO inhibition (e.g., by use of a suitable 
prodrug or formulation). 
In view of the foregoing, it is preferable to administer deprenyl compounds 
by a route that minimizes metabolism to inhibitor compounds such as 
(-)-methamphetamine and (-)-amphetamine, while allowing metabolism to 
active compounds such as (-)-desmethyldeprenyl. Metabolism to an active 
compound can occur at the desired site of activity, e.g., in the optic 
nerve. Thus, prodrugs, which are metabolized to active compounds, are 
useful in the methods of the invention. 
It has been found that certain deprenyl compounds have greater therapeutic 
efficacy (e.g., are effective at lower doses) when administered so as to 
decrease or prevent the "first-pass" effect. Accordingly, intraperitoneal 
or especially subcutaneous injection are preferred routes of 
administration. In preferred embodiments, a deprenyl compound is 
administered in divided doses. For example, a deprenyl compound can be 
administered by frequent (e.g., pulsed) injections, or by a controlled 
infusion, which can be constant or programmably varied as described above. 
In preferred embodiments in which a deprenyl compound is administered 
orally, the deprenyl compound can be formulated to reduce the amount of 
hepatic metabolism after oral administration and thereby improve the 
therapeutic efficacy. 
In certain embodiments, the deprenyl compounds of the invention can be 
formulated to ensure proper distribution in vivo. For example, the 
blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To 
ensure that the therapeutic compounds of the invention cross the BBB (if 
desired), they can be formulated, for example, in liposomes. For methods 
of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 
5,374,548; and 5,399,331. The liposomes may comprise one or more moieties 
which are selectively transported into specific cells or organs 
("targeting moieties"), thus providing targeted drug delivery (see, e.g., 
V. V. Ranade (1989) J Clin. Pharmacol. 29:685). Exemplary targeting 
moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to 
Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. 
Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 
357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); 
surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 
1233:134); gp120 (Schreier et al. (1994)J. Biol. Chem. 269:9090); see also 
K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. 
J. Fidler (1994) Immunomethods 4:273. In a preferred embodiment, the 
therapeutic compounds of the invention are formulated in liposomes; in a 
more preferred embodiment, the liposomes include a targeting moiety. 
The following invention is further illustrated by the following example, 
which should in no way be construed as being further limiting. The 
contents of all references, pending patent applications and published 
patent applications, cited throughout this application are hereby 
incorporated by reference. It should be understood that the animal model 
for glaucoma and optical nerve rescue used in the example is accepted and 
that a demonstration of efficacy in these models is predictive of efficacy 
in humans. 
EXAMPLES 
Example 
METHODS 
All animals were handled according to the Declaration of Helsinki and The 
Guiding Principles in the Care and Use of Animals. Under halothane/nitrous 
oxide anaesthesia, two groups of adult Strague-Dawley rats weighing 
200-300 gm, eight in each group, were placed in a stereotactic instrument 
and 1.0 micoliter of 3% Fluoro-Gold (FG, Fluorochrome Inc.), a retrograde 
tracer, was stereotactically injected bilaterally near to the center of 
each superior colliculus (Bregma co-ordinates--5.3 mm, 2 mm lateral and 
4.5 mm deep). The injections were delivered from a 10 microliter Hamilton 
syringe over a period of fifteen minutes. Slow injections were used to 
avoid tissue disruption and the large volume was employed to insure 
diffusion would extend over the full length of each superior colliculus 
(SC) (see Tatton et al. ((1991) Neurosc. Letters 131, 179-182) for details 
and rationale of FG injection to pre-label the cell bodies of neurons 
sending axons to a specific Brian structure). 
It has been reported that 40-50% of the neurons whose cell bodies are 
located in the retinal ganglion cell layer (RGCL) of the rat send an axon 
to the optic nerve and are RGCs (Cowey et al. (1979) Exp. Brain Res. 35, 
457-64; Perry V. H. (1981) Neuroscience 6, 931-44: and Linden et al. 
(1989) Brain Res. 272, 145-149). The remaining cell bodies in the rat RGCL 
have been shown to be displaced amacrine cells which do not project to the 
optic nerve (Perry V. H. (1981) Neuroscience 6, 931-44). Most RGCs (&gt;95%) 
in rats send their axons to the SC with as many as 10% also sending 
branches to the lateral geniculate body (Cowey et al. (1979) Exp. Brain 
Res. 35, 457-64). Hence the FG injections into the superior colliculus 
would be expected to retrogradely-label the cell bodies of RGCs projecting 
to the superior colliculus (RGC.sup.SC s) but would not label the 
displaced amacrines in RGCL thereby providing an unambiguous method of 
RGC.sup.SC identification. 
Four days were allowed for the retrograde transport of the FG to the 
RGC.sup.SC s cell bodies and then the left optic nerve of the rats was 
exposed and crushed by applying the tips of Dumont number 5 forceps to the 
nerve immediately behind the globe for ten seconds. Careful attention was 
used to avoid the central retinal artery. Each nerve was crushed three 
times at the same site. Following the crushes, the nerve was examined with 
the aid of an operating microscope to ensure that the axonal component of 
the nerve was divided into two clearly separated stumps surrounded by an 
unbroken dural sheath. Retinal artery patency was confirmed by direct 
ophthalmoscopy. 
One group of eight rats received (-)-deprenyl (1 mg/kg) by intraperitoneal 
(IP) injection every two days for a fourteen day period beginning at the 
time of optic nerve crush. The remaining group of eight rats received IP 
injections of saline on the same schedule. Fourteen days after the optic 
nerve crush all sixteen rats were euthanized with an overdose of somnotol 
and perfused transcardially with 4% paraformaldehyde in phosphate buffer. 
The brains and retinas were immersed in 20% sucrose for twenty four hours 
and then frozen in -70 degrees C. methylbutane. Serial 10 mm sections were 
cut through the upper brain stem and diencephalon of the animals and 
viewed under fluorescence microscopy to determine the location and extent 
of the stereotactic injections of FG. Serial 5 mm sections of the retinas 
were cut and every third section was Nissl stained. In order to detect 
even low level FG fluorescence in neuronal cell bodies or processes, the 
FG labelled brain sections and the retinal sections were examined with the 
aid of a Hamarmatsu intensification camera that allowed the fluorescent 
images to be digitized using a Matrox frame grabber controlled by image 
analysis software (Universal Metamorph). This allowed for the creation of 
computer images of neuronal cell bodies containing FG in the sections 
taken from areas of the stereotactic injections and sections taken through 
each of the retinas. 
About 25 sections were chosen randomly from the serial sections taken from 
each retina. Sections were only chosen from the middle 70% of the serial 
sections where section length exceeded 6 mm. Nissl stained cell bodies in 
the RGCL were counted at 1000X magnification under oil immersion on a 
Leitz Orthoplan microscope. RGCL somata were only counted if they 
contained a well defined nucleus. The cross sectional length of the 
ganglion cell layer for each section was measured by transferring the 
retinal section image into a computer using a CCD camera (Dage Ltd.) and 
IPPLUS 2.1 software (Media Cybernetics). The number of RGCL neuronal cell 
bodies (RGCLncbs) per mm length of retinal crossection was calculated and 
were pooled for each of the four lesion/treatment groups 
(uncrushed-saline, uncrushed-deprenyl, crushed-saline and 
crushed-deprenyl). 
A similar group of sections were randomly chosen from one of the two 
remaining series of retinal sections. Those sections were lightly Nissl 
stained and then were mounted in Fluoromount to allow for counts of Nissl 
stained RGCLncbs under brightfield microscopy and counts of RGCLncbs 
containing FG under fluorescence microscopy from identical retinal fields. 
Determination of numbers of RGCLncbs that met the Nissl criteria for 
neurons and also contained FG were made by switching back and forth from 
brightfield to fluorescence microscopy. The ratio of the FG containing 
cell bodies/Nissl stained neuronal somata in each section was used to 
determine the proportion of RGCLncbs that were RGC.sup.SC s. 
Previous studies have shown that the density of RGCs varies from 1600 per 
mm.sup.2 in the peripheral retina to 2500 per mm.sup.2 in some parts near 
the area centralis (Perry V. H. (1981) Neuroscience 6, 931-44). Since the 
sections that were counted spanned 70% of the width of each retina, they 
would be expected to include different proportions of the central and 
peripheral retina. This would result in widely varying RGCL ncbs/mm for 
different Nissl stained sections and marked differences in the numbers of 
Nissl stained and FG containing cell bodies that would be identified as 
RGC.sup.SC s. Accordingly, the distributions of cell body counts were 
determined from the pooled values for each experimental group. To 
determine the statistical significance of any changes in those 
distributions, the count for each section was treated as a single value 
and the Kolmogorov-Sminov test (Siegel S. Non-parametric statistics for 
the behavioral sciences. In: New York: McGraw Hill Book Company. 
1956:127-136) was used to compare the values obtained from the four 
different experimental groups in a pairwise fashion. The 
Kolmogorov-Smirnov test is a non-parametric test which does not assume an 
underlying binomial distribution or that the values are linearly related 
to each other. The method is optimal for comparing widely distributed data 
(see Ju et al. (Exp. Neurol. 126, 233-246) for an example of its use to 
determine the significance of changes of pooled values taken from large 
numbers of microscopic sections). 
An intensified fluorescent image of a frontal section taken through the SC 
at 18 days after an injection of FG was examined. Cell bodies and 
processes in all layers of the SC were found to be labelled with FG. 
Because of the relatively large injection, FG labelling could be seen over 
the full rostrocaudal length of both SCs and extended to nearby brainstem 
and diencephalic structures, including the most caudal portions of the 
lateral geniculate body. This demonstrated that most, if not all, RGC 
axons projecting to the SC had taken up FG and transported the fluorescent 
marker to their retinal cell bodies. Photomicrographs of the same field of 
a single retinal section viewed alternately under interference contrast 
microscopy and fluorescence microscopy showed typical RGC.sup.SC s that 
were Nissl stained and fluoresced for FG. 
FIG. 1 presents box plots of the distributions of the counts of RGCLncbs 
for the four experimental groups presented with scales showing the counts 
per section or the percent of cell bodies relative to the mean value found 
in the uncrushed saline treated group. There was no statistical difference 
between the distributions for uncrushed saline (48.35.+-.10.75 /mm) and 
the uncrushed deprenyl (48.39.+-.8.48 /mm) groups (p&gt;0.05). The 
distribution for the uncrushed saline group was significantly different 
from the distribution for the crushed saline group (22.70.+-.7.44/mm, 
p&lt;0.0001) and the distribution for the crushed deprenyl group 
(32.60.+-.9.94, p&lt;0.01). Importantly, the distribution for the crushed 
deprenyl group was shifted to significantly greater values than that for 
the crushed saline group (p&lt;0.001). In short, the numbers of neuronal cell 
bodies/mm in the RGCL of crushed saline retinas were reduced to an average 
of 46% of those in uncrushed saline retinae and (-)-deprenyl treatment 
increased that value to an average of 65%. If, as previously reported, 
40-50% of the neurons in the rat RGCL do not send axons to the optic nerve 
and would not be damaged by the crushes, then these values indicate less 
than 10% survival for RGCs at fourteen days in the crushed saline retinas 
and approximately 30-40% survival in the crushed deprenyl retinas. 
To determine whether the joint counts of Nissl and FG cell bodies in the 
RGCL was a reliable estimate of the proportion of RGCs in the layer, the 
counts of Nissl stained cell bodies and FG labelled cell bodies were 
plotted as x and y values for varying lengths of retinal sections taken 
from different portions of the differently lesioned and treated retinas 
(FIG. 2). The Nissl and FG counts were found to covary linearly 
independently of section length or location for the uncrushed saline, 
uncrushed deprenyl, and crushed deprenyl retinal groups with regression 
coefficients 0.98, 0.96 and 0.90 respectively and y axis intercepts near 
zero. The slopes did not differ significantly (p&gt;0.05) for the uncrushed 
saline (0.478.+-.0.021) and uncrushed deprenyl retinal groups 
(0.379.+-.0.021). The slope of the crushed deprenyl groups 
(0.219.+-.0.019) was significantly different from the uncrushed saline and 
uncrushed deprenyl groups (p's&lt;0.0001). Since the counts of FG labelled 
cell bodies in the crushed saline retinas was very low (average of 
0.66/mm), fitting the data to a linear relationship was not valid. 
The mean ratios of FG labelled to Nissl stained somata was 0.425.+-.0.045 
for the uncrushed saline group, 0.388.+-.0.053 for the uncrushed deprenyl 
group, 0.003.+-.0.01 for the crushed saline group, and 0.245.+-.0.055 for 
the crushed deprenyl group. Therefore, by pooling the uncrushed saline and 
uncrushed deprenyl results, these data estimate that approximately 40.7% 
of the neuronal cell bodies in the RGCL of retinas with uncrushed optic 
nerves sent their axons to the SC. These results are consistent with 
previous reports that 40-50% of the neurons in the rat RGCL are RGCs which 
send an axon to the optic nerve and that greater than 95% of rat RGCs send 
axons to the SC. 
The counts of RGCLncbs and the proportions of FG-labelled cell bodies were 
combined to determine the distributions RGC.sup.SC s expressed as 
percentages of the mean value of the distribution for the uncrushed saline 
retinas (FIG. 3). There were no significant differences (p&gt;0.05) in the 
distributions for the uncrushed saline RGC.sup.SC group (100.0.+-.22.2%) 
and the uncrushed (-)-deprenyl RGC.sup.SC group (87.0.+-.15.2%). In 
contrast, crushed saline RGC.sup.SC group (3.0.+-.1.0%, p&lt;0.0001) and the 
crushed (-)-deprenyl RGC.sup.SC group (36.9.+-.11.2%, p&lt;0.0001) were 
distributed differently than the uncrushed saline RGC.sup.SC group. The 
increase in survival in the crushed (-)-deprenyl RGC.sup.SC group was 
significantly different from the crushed saline RGC.sup.SC group 
(p&lt;0.001). 
EQUIVALENTS 
Those skilled in the art will recognize or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.