The present invention includes phosphoramidate derivatives, compositions containing the same, and methods of using them as NAALADase inhibitors, particularly for the treatment of glutamate abnormalities and associated nervous tissue insult in a animal and for treatment of prostate disease and prostate cancer by inhibition of NAALADase enzyme.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to novel compounds and compositions which 
inhibit N-Acetylated .alpha.-Linked Acidic Dipeptidase (NAALADase) enzyme 
activity, and in particular to phosphoramidate derivatives and 
compositions containing the same which inhibit NAALADase enzyme activity 
in humans and warm-blooded animals. The present invention is also directed 
to novel compounds and compositions which inhibit NAALADase enzyme 
activity and are useful as novel agents for treatment of glutamate 
abnormalities in animals, particularly the prevention or alleviation of 
brain damage caused by strokes and other types of ischemic damage. 
Furthermore, the present invention is also directed to novel compounds and 
compositions useful as novel agents for treatment of cancer and related 
diseases of the prostate, particularly prostate cancer. 
2. Description of the Prior Art 
Ischemia 
Ischemia, a localized tissue anemia resulting from the obstruction of the 
inflow of arterial blood, can cause extensive damage when it occurs in the 
brain or central nervous system. Central nervous tissue, and to a lesser 
extent peripheral nervous tissue, has poor reparative abilities. Thus 
damage to nervous tissue causes significant permanent disability and is a 
frequent cause of death. Damage to nervous tissue may occur in many ways, 
not only through ischemia in cerebrovascular accidents, but also in 
cerebral circulatory disturbances, episodes of absolute and relative 
hypoxia, metabolic disturbances and various forms of trauma. 
Global ischemia occurs under conditions in which blood flow to the entire 
brain ceases for a period of time, such as may result from cardiac arrest. 
Focal ischemia occurs under conditions in which a portion of the brain is 
deprived of its normal blood supply, such as may result from 
thromboembolytic occlusion of a cerebral vessel, traumatic head injury, 
edema, and brain tumors. In areas of focal ischemia or damage, there is a 
core of more profound damage, surrounded by a perifocal penumbra of lesser 
damage. This is because the neurons in the penumbra can for a time 
maintain homeostasis thus rendering them potentially more salvageable by 
pharmacological agents. 
Both global and focal ischemic conditions have the potential for producing 
widespread neuronal damage, even if the ischemic condition is transient. 
Although some permanent neuronal injury may occur in the initial mixture 
following cessation of blood flow to the brain, the damage in global and 
focal ischemia occurs over hours or even days following the ischemic 
onset. Much of this neuronal damage is attributed to glutamate toxicity 
and secondary consequences of reperfusion of the tissue, such as the 
release of vasoactive products by damaged endothelium, and the release by 
the damaged tissues of cytotoxic products including free radicals, 
leukotrienes, and the like. 
Glutamate neurotoxicity, which is a major factor in ischemic neuronal 
injury, appears to begin with resumption of oxidative metabolism and thus 
occurs both during reversible ischemia and during recovery. Many attempts 
have been made to avoid this problem by blocking of the various receptors 
including NMDA receptors, AMPA receptors, Kainate receptors, and MGR 
receptors, which are stimulated by glutamate and are also strongly 
involved in nerve cell death occurring after the onset of global or focal 
ischemia. When ischemia occurs, such as during a stroke or heart attack, 
there is an excessive release of endogenous glutamate, resulting in the 
overstimulation of NMDA receptors, AMPA receptors, Kainate receptors, and 
MGR receptors. Interaction of the glutamate with these receptors causes 
the ion channel associated with these receptors to open, allowing a flow 
of cations across the cell membrane. This flux of ions, particularly 
Ca.sup.2+ into the cells, plays an important role in nerve cell death. 
Much activity has been undertaken in attempting to prevent glutamate from 
exciting these receptors. This has proven difficult since these receptors 
each have many different sites to which the glutamate may bind. 
Furthermore, many of the compositions that are effective in blocking 
glutamate from these receptors have also proven in clinical trials to be 
toxic to the animal that they are administered to. 
Currently there is no known effective treatment for nervous tissue damage. 
At best, supportive measures may be taken in a hospital during the period 
after nervous tissue insult, such as stroke or trauma. Several drug 
strategies that have been proposed for treatment of stroke and other 
neuronal conditions related to ischemia have met with differing and 
incomplete success as agents to protect the nervous system from damage. 
Anti-coagulants, such as heparin, have been examined, but with mixed 
results. Similarly, antivasoconstriction agents, such as flunarazine, 
excitatory neurotransmitter antagonists, such as MK-801 and AP7, and 
anti-edemic compounds have shown mixed results, with no clear benefits to 
outweigh a variety of side effects, including neurotoxicity or increased 
susceptibility to infection. Nimodipine, a calcium channel blocker, is 
used clinically to treat vasospasm after subarachnoid hemorrhage. 
Methylprednisolone, a steroid, in very high doses is helpful in spinal 
cord compression. Tirilazad, a 21-aminosteroid linked to a free radical 
scavenger, underwent clinical trials to decrease the damage caused by 
stroke. 
The high rate of disability from nervous insults demonstrates the need for 
an effective neuroprotective agent. Unfortunately, drugs which have been 
proposed to date for the treatment of stroke and other ischemic-related 
conditions of the brain are either (i) relatively ineffective, (ii) 
effective only at dosage levels where undesired side effects are observed, 
(iii) produce systemic effects, such as hypotension, which comprise the 
potential effectiveness of the drug, and/or (iv) are toxic to the patient. 
Glutamate Toxicity Within the Central Nervous System 
Efforts to examine the role of glutamate toxicity in diseases of the brain, 
i.e. epilepsy, amyotrophic lateral sclerosis (ALS), schizophrenia, and 
Alzheimer's disease, led researchers in an attempt to ascertain the exact 
role of N-acetylated .alpha.-linked acidic dipeptidase (NAALADase) and 
N-acetyl-L-aspartate-L-glutamate (NAAG) in the central nervous system 
(CNS). 
The dipeptide NAAG is an abundant nervous system specific peptide which is 
present in synaptic vesicles and released upon neuronal stimulation in 
several systems. As a major peptidic component of the brain, NAAG is 
present in levels comparable to that of the major inhibitory 
neurotransmitter .gamma.-aminobutyric acid (GABA). Although NAAG was first 
isolated in 1964, there was little activity toward elucidating its role in 
the CNS until the deleterious nature of excess glutamate in a variety of 
disease states became apparent. Due to its structural similarity to 
glutamate, NAAG has been suggested to have a variety of roles similar to 
those of glutamate itself, including functioning as a neurotransmitter or 
a cotransmitter, neuromodulator, or as a precursor of the neurotransmitter 
glutamate. NAAG has elicited excitatory responses both in vitro and in 
vivo, but is significantly less potent than glutamate. 
Prostate Cancer 
In a separate area of research, prostate cancer has been determined to now 
be the leading form of cancer among men and the second most frequent cause 
of death from cancer in men. It is estimated that more than 165,000 new 
cases of prostate cancer were diagnosed in 1993, and more than 35,000 men 
died from prostate cancer in that year. Additionally, the incidence of 
prostate cancer has increased by 50% since 1981, and mortality from this 
disease has continued to increase. Previously, most men died of other 
illnesses or diseases before dying from their prostate cancer. We now face 
increasing morbidity from prostate cancer as men live longer and the 
disease has the opportunity to progress. 
Current therapies for prostate cancer focus exclusively upon reducing 
levels of dihydrotestosterone to decrease or prevent growth of prostate 
cancer. In addition to the use of digital rectal examination and 
transrectal ultrasonography, prostate-specific antigen (PSA) concentration 
is frequently used in the diagnosis of prostate cancer. 
Prostate Specific Antigen (PSA) is a well known prostate cancer marker. PSA 
is a protein produced by prostate cells and is frequently present at 
elevated levels in the blood of men who have prostate cancer. PSA has been 
shown to correlate with tumor burden, serve as an indicator of metastatic 
involvement, and provide a parameter for following the response to 
surgery, irradiation, and androgen replacement therapy in prostate cancer 
patients. It should be noted that Prostate Specific Antigen (PSA) is a 
completely different protein from Prostate Specific Membrane Antigen 
(PSMA). Although they have similar nomenclature, the two proteins have 
different structures and functions. 
Prostate Specific Membrane Antigen (PSMA) 
In 1993, the molecular cloning of a prostate-specific membrane antigen 
(PSMA) was reported as a potential prostate carcinoma marker and 
hypothesized to serve as a target for imaging and cytotoxic treatment 
modalities for prostate cancer. Antibodies against PSMA have been 
described and examined clinically for diagnosis and treatment of prostate 
cancer. In particular, Indium-111 labelled PSMA antibodies have been 
described and examined for diagnosis of prostate cancer and 
itrium-labelled PSMA antibodies have been described and examined for the 
treatment of prostate cancer. 
PSMA is expressed in prostatic ductal epithelium and is present in seminal 
plasma, prostatic fluid and urine. In 1996, it was found that the 
expression of PSMA cDNA actually confers the activity of NAALADase. This 
is entirely unexpected because until recently NAALADase research has been 
limited to its role in the brain and its effect on neurotransmitters 
whereas PSMA has been described and examined for the diagnosis and therapy 
of prostate cancer. 
NAALADase 
In 1988, a brain enzyme, NAALADase, was identified which hydrolyzes NAAG to 
N-acetylaspartate (NAA) and glutamate (See TABLE I, below). 
Catabolism of NAAG by the peptidase NAALADase. 
TABLE 1 
______________________________________ 
##STR1## 
##STR2## 
______________________________________ 
NAALADase, which derives its name from its structural specificity for 
N-acetylated acidic dipeptides, is a membrane-bound metallopeptidase 
having a denatured molecular mass of 94 kDa, that catabolizes NAAG to 
N-acetylaspartate (NAA) and glutamate. It has been demonstrated that 
.sup.3 H!NAAG is degraded in vivo by an enzyme with the pharmacological 
characteristics of NAALADase, which supports a role for NAALADase in the 
metabolism of endogenous NAAG. 
Rat NAALADase activity has been extensively characterized and demonstrates 
a high affinity for hydrolysis of its putative substrate NAAG, with a 
Km=140 nM. Recently, NAALADase also has been shown to cleave the 
non-acetylated peptide, aspartylglutamate, with high affinity. Research 
has also found that the enzyme is membrane-bound, stimulated by chloride 
ions, and inhibited by polyvalent cation chelators, suggesting that it is 
a metallopeptidase. 
In mammals, NAALADase is enriched in synaptic plasma membranes and is 
primarily localized to neural tissue and the kidneys. NAALADase has not 
been found in large quantities in the mammalian liver, heart, pancreas, or 
spleen. 
Examination of NAAG and NAALADase has been conducted for several different 
human and animal pathological conditions. It has been demonstrated that 
intra-hippocampal injections of NAAG elicit prolonged seizure activity. 
More recently, it was reported that rats genetically prone to epileptic 
seizures have a persistent increase in their basal level of NAALADase 
activity. These observations are consistent with the hypothesis that 
increased availability of synaptic glutamate elevates seizure 
susceptibility, and suggest that NAALADase inhibitors may provide 
anti-epileptic activity. 
NAAG and NAALADase have also been implicated in the pathogenesis of ALS and 
in the pathologically similar animal disease called Hereditary Canine 
Spinal Muscular Atrophy (HCSMA). It has been shown that concentrations of 
NAAG and its metabolites--NAA, glutamate and aspartate--are elevated two- 
to three-fold in the cerebrospinal fluid of ALS patients and HCSMA dogs. 
In addition, NAALADase activity is significantly increased (two- to 
three-fold) in post-mortem spinal cord tissue from ALS patients and HCSMA 
dogs. Although highly speculative, NAALADase inhibitors may be clinically 
useful in curbing the progression of ALS if increased metabolism of NAAG 
is responsible for the alterations of CSF levels of these acidic amino 
acids and peptides. Abnormalities in NAAG levels and NAALADase activity 
have also been documented in post-mortem schizophrenic brain, specifically 
in the prefrontal and limbic brain regions, underscoring the importance of 
examining the metabolism of NAAG in the pathophysiology of schizophrenia. 
The identification and purification of NAALADase led to the proposal of 
another role for NAAG: specifically that the dipeptide may serve as a 
storage form of synaptic glutamate. 
NAALADase Inhibitors 
Only a few NAALADase inhibitors have been identified in the prior art thus 
far and those that have been identified have only been used in 
non-clinical neurological research. Examples of such inhibitors include 
general metallopeptidase inhibitors such as o-phenanthrolene, metal 
chelators such as EGTA and EDTA, and peptide analogs such as quisqualic 
acid and beta-NAAG. It should be noted that prior to the compositions of 
the present invention, NAALADase inhibitors have either had toxic side 
effects or were not capable of being administered in pharmaceutically 
effective amounts. 
SUMMARY OF THE INVENTION 
The present invention is directed to novel compounds and compositions which 
inhibit N-Acetylated .alpha.-Linked Acidic Dipeptidase (NAALADase) enzyme 
activity, and in particular to phosphoramidate derivatives and 
compositions containing the same which inhibit NAALADase enzyme activity 
in humans and warm-blooded animals. The present invention is also directed 
to novel compounds and compositions which inhibit NAALADase enzyme 
activity and are useful as novel agents for treatment of glutamate 
abnormalities in animals, particularly the prevention or alleviation of 
brain damage caused by strokes and other types of ischemic damage. 
The present invention is also directed to the surprising discovery that 
NAALADase inhibitors exhibit a significant inhibitory effect on the growth 
of cancer cells, and particularly prostate cancer cells. The present 
disclosure relates to novel compositions containing dipeptidase 
inhibitors, and more particularly, to compounds and compositions which 
inhibit N-Acetylated .alpha.-Linked Acidic Dipeptidase (NAALADase) enzyme 
activity useful for treatment for diseases of the prostate, particularly, 
prostate cancer. Furthermore, as has been found in other tissues of the 
body, NAALADase inhibitors may show efficacy in the treatment of other 
forms of cancer. 
The present invention is based upon the surprising discovery that the 
NAALADase inhibitors of the present invention exhibit increased potency 
over prior art compounds. 
Preferred compositions of the present invention include compounds having 
the following formula: 
##STR3## 
where R and R1 are independently hydrogen, C.sub.1 -C.sub.9 straight or 
branched chain alkyl or alkenyl group, C.sub.3 -C.sub.8 cycloalkyl, 
C.sub.3 or C.sub.5 cycloalkyl, C.sub.5 -C.sub.7 cycloalkenyl, or Ar.sub.1. 
The present invention also contemplates the use of said alkyl, alkenyl, 
cycloalkyl, cycloalkenyl or aryl groups to be optionally substituted with 
C.sub.3 -C.sub.8 cycloalkyl, C.sub.3 or C.sub.5 cycloalkyl, C.sub.5 
-C.sub.7 cycloalkenyl, C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4 alkenyl, 
hydroxy, halo, hydroxyl, nitro, trifluoromethyl, C.sub.1 -C.sub.6 straight 
or branched chain alkyl or alkenyl, C.sub.1 -C.sub.4 alkoxy, C.sub.1 
-C.sub.4 alkenyloxy, phenoxy, benzyloxy, amino, or Ar.sub.1, and where 
Ar.sub.1 is selected from the group consisting of 1-napthyl, 2-napthyl, 
2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-, 3-, or 
4-pyridyl, or phenyl, having one to three substituents which are 
independently selected from the group consisting of hydrogen, halo, 
hydroxyl, nitro, trifluoromethyl, C.sub.1 -C.sub.6 straight or branched 
alkyl or alkenyl, C.sub.1 -C.sub.4 alkoxy or C.sub.1 -C.sub.4 alkenyloxy, 
phenoxy, benzyloxy, and amino; or pharmaceutically acceptable salts or 
hydrates thereof. 
Especially preferred compounds of the present invention are selected from 
the group consisting of: 
N-Methylhydroxyphosphinyl!glutamic Acid; 
N-Ethylhydroxyphosphinyl!glutamic Acid; 
N-Propylhydroxyphosphinyl!glutamic Acid; 
N-Butylhydroxyphosphinyl!glutamic Acid; 
N-Phenylhydroxyphosphinyl!glutamic Acid; 
N-(Phenylmethyl)hydroxyphosphinyl!glutamic Acid; 
N-((2-Phenylethyl)methyl)hydroxyphosphinyl!glutamic Acid; and 
N-Methyl-N-Phenylhydroxyphosphinyl!glutamic Acid. 
Compounds of the present invention which are highly preferred are selected 
from the group consisting of: 
N-Propylhydroxyphosphinyl!glutamic Acid; 
N-Butylhydroxyphosphinyl!glutamic Acid; 
N-Phenylhydroxyphosphinyl!glutamic Acid; and 
N-(Phenylmethyl)hydroxyphosphinyl!glutamic Acid. 
Compositions within the scope of the present invention contain the above 
described compounds and are formulated with a suitable pharmaceutical 
carrier. Such carriers are especially formulated in order to best utilize 
the compound for the particular treatment, such as for treating glutamate 
abnormalities in an animal, treating nervous insult as defined herein, 
treating ischemia in an animal, treatment using the compounds of the 
present invention as glutamate modulators, treatment to provide recovery 
of tissues after an ischemic event, and treatment to decrease injuries 
caused by ischemia such as brain injuries caused by global ischemia or 
focal ischemia. 
Further preferred embodiments include the use of additional therapeutic 
agents useful for treating ischemia. The agent can also include any 
pharmaceutical compound useful for the treatments described herein to be 
delivered in combination with the compounds and compositions of the 
present invention. 
Compositions within the scope of the present invention also are formulated 
with a suitable pharmaceutical carrier in order to best utilize the 
compound for inhibition of tumor growth, inhibition of tumor cell growth, 
and inhibition of NAALADase enzyme activity. A particularly preferred 
tumor type is prostatic adenocarcinoma. 
Yet another preferred embodiment is directed to a composition for treating 
prostate diseases selected from the group consisting of prostate cancer 
and benign prostatic hyperplasia in an animal, which comprises: (i) the 
compound described above and (ii) a pharmaceutically acceptable carrier 
for administering said compound to said animal. 
Further preferred embodiments include the use of additional therapeutic 
agents useful for treating diseases of the prostate. Such agents may be 
selected from the group consisting of: therapeutic hormones, 
chemotherapeutic agents, monoclonal antibodies, anti-angiogenesis agents, 
and radiolabelled compounds. The agent can also include any pharmaceutical 
compound useful for the treatments described herein to be delivered in 
combination with the compounds and compositions of the present invention. 
The methods of the present invention include using the compounds of the 
present invention and/or compositions containing phosphoramidate 
derivatives that inhibit NAALADase enzyme activity which have been found 
useful for the treatment of NAALADase related indications. Especially 
preferred indication include treating glutamate abnormalities in an 
animal, treating nervous insult as defined herein, treating ischemia in an 
animal, treatment using the compounds of the present invention as 
glutamate modulators, treatment to provide recovery of tissues after an 
ischemic event, and treatment to decrease injuries caused by ischemia such 
as brain injuries caused by global ischemia or focal ischemia. It is 
contemplated that these methods can also utilize additional therapeutic 
agents useful for treating nerve-related indications such as ischemia. 
Such additional agents are known to persons of ordinary skill in the art. 
Further preferred methods of the present invention include treatment using 
the compounds and compositions described herein for inhibition of tumor 
growth, inhibition of tumor cell growth, and inhibition of NAALADase 
enzyme activity. A particularly preferred tumor type is prostatic 
adenocarcinoma. 
Additional preferred embodiments are directed to compounds and compositions 
for treating prostate diseases selected from the group consisting of 
prostate cancer and benign prostatic hyperplasia in an animal.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to novel NAALADase inhibitors. The 
compounds of the present invention also exhibit increased potency due to 
their novel structures. The compounds of the present invention are useful 
for treating ischemia, in particular global and focal ischemia, in humans 
and warm-blooded animals. 
Furthermore, the compounds of the present invention include phosphoramidate 
derivatives that inhibit NAALADase enzyme activity and which have been 
found useful for inhibiting the growth of prostate cancer cells. Although 
the use of NAALADase inhibitors is exemplified for the treatment of 
prostate cancer, compositions including NAALADase inhibitors are not 
limited to this specific form as cancer. NAALADase has been found in other 
tissues in the body, which results in efficacy in the treatment of other 
forms of cancer. For example, the kidney, brain, and testis have NAALADase 
present. As a result, in these examples brain cancer, kidney cancer, or 
testicular cancer may be treated via this approach. Other tissues will 
correlate with other treatments accordingly. 
NAALADase is an enzyme which is a membrane-bound metalloprotease that 
hydrolyzes the dipeptide, N-acetyl-L-aspartate-L-glutamate (NAAG) to yield 
glutamate and N-acetylaspartate. The amino acid L-glutamate is a 
neurotransmitter that mediates fast neuronal excitation in a majority of 
synapses in the central nervous system (CNS). Once released into the 
synapse, L-glutamate can stimulate the N-methyl-D-aspartate (NMDA) 
receptor, a subtype of an excitatory amino acid receptor. It has been 
discovered that excessive activation of the NMDA receptor has been 
implicated in a variety of acute as well as chronic neurophatholgical 
processes such as ischemia, epilepsy and Huntington's disease. Thus, 
considerable effort has focused on finding novel therapeutic agents to 
antagonize the postsynaptic effects of L-glutamate mediated through the 
NMDA receptor. Although not limited to any one particular theory, it is 
believed that the compounds of the present invention modulate levels of 
glutamate by acting on a storage form of glutamate which is hypothesized 
to be upstream from the effects mediated by the the NMDA receptor. 
Preferred compositions of the present invention include compounds having 
the following formula: 
##STR4## 
where R and R1 are independently hydrogen, C.sub.1 -C.sub.9 straight or 
branched chain alkyl or alkenyl group, C.sub.3 -C.sub.8 cycloalkyl, 
C.sub.3 or C.sub.5 cycloalkyl, C.sub.5 -C.sub.7 cycloalkenyl, or Ar.sub.1. 
The present invention also contemplates compounds wherein said alkyl, 
alkenyl, cycloalkyl, cycloalkenyl, or aryl groups to be optionally 
substituted with C.sub.3 -C.sub.8 cycloalkyl, C.sub.3 or C.sub.5 
cycloalkyl, C.sub.5 -C.sub.7 cycloalkenyl, C.sub.1 -C.sub.4 alkyl, C.sub.1 
-C.sub.4 alkenyl, hydroxy, halo, hydroxyl, nitro, trifluoromethyl, C.sub.1 
-C.sub.6 straight or branched chain alkyl or alkenyl, C.sub.1 -C.sub.4 
alkoxy, C.sub.1 -C.sub.4 alkenyloxy, phenoxy, benzyloxy, amino, or 
Ar.sub.1, and where Ar.sub.1 is selected from the group consisting of 
1-napthyl, 2-napthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thienyl, 
3-thienyl, 2-, 3-, or 4-pyridyl, or phenyl, having one to three 
substituents which are independently selected from the group consisting of 
hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C.sub.1 -C.sub.6 
straight or branched alkyl or alkenyl, C.sub.1 -C.sub.4 alkoxy or C.sub.1 
-C.sub.4 alkenyloxy, phenoxy, benzyloxy, and amino; or pharmaceutically 
acceptable salts or hydrates thereof. 
It has been unexpectedly found that the right hand side of the molecular 
structure depicted above is necessary for substrate recognition by 
NAALADase enzyme. Thus, the present invention only contemplates 
substitutions to the left hand side and at the nitrogen, indicated by the 
R and R1 group, of the phosphoramidate derivative structure above. 
DEFINITIONS 
"NAALADase" as used herein refers to N-Acetylated Alpha-Linked Acidic 
Dipeptidase. The enzyme was originally named for it's substrate 
specificity for hydrolyzing N-acetylated alpha-linked acidic dipeptides. 
Currently, it is known that the enzyme has a broader range of substrate 
specificity than originally discovered, particularly that the enzyme does 
not require N-acetylation or alpha-linkage. Thus, as used herein 
"NAALADase" encompasses other names used in the literature such as NAAG 
hydrolyzing enzyme and NAALA dipeptidase. 
As used in the specification and claims, the chemical structures refer to 
conventional designations. For example, "alkyl" is a paraffinic 
hydrocarbon group which may be derived from an alkane by dropping one 
hydrogen from the formula, such as methyl, ethyl, propyl, isopropyl, 
butyl, and so forth. "Alkenyl" is an olefinic unsaturated hydrocarbon 
having one or more double bonds such as methylene, ethylene, propylene, 
isopropylene, butylene, and so forth. The term "Cyclo", used herein as a 
prefix, refers to a structure characterized by a closed ring. The term 
"oxy", used herein as a suffix, i.e. alkoxy, alkenoxy, phenoxy, and so 
forth, refers to having one or more oxygen molecules attached. Thus, the 
term "carboxy" may describe, for example, a carbon having both an oxygen 
and a hydroxy moiety attached. 
"Halogen" includes bromo, fluoro, chloro and iodo; "halomethyl" includes 
mono-, di-, and tri-halo groups including trifluoromethyl; amino compounds 
include amine (NH.sub.2) as well as substituted amino groups comprising 
alkyls of one through six carbons; "Ar.sub.1 ", chemical shorthand for 
"aryl", includes aromatic ring compounds such as benzene, phenyl, 
naphthyl, indolyl, furyl, thienyl, pyridyl, and substituted forms thereof; 
"aralkyl", is an aryl being attached through an alkyl chain, straight or 
branched, of from one through six carbons such as phenylpropyl group. 
The term "inhibition", in the context of enzyme inhibition, relates to 
reversible enzyme inhibition such as competitive, uncompetitive, and 
noncompetitive inhibition. This can be experimentally distinguished by the 
effects of the inhibitor on the reaction kinetics of the enzyme, which may 
be analyzed in terms of the basic Michaelis-Menten rate equation. 
Competitive inhibition occurs when the inhibitor can combine with the free 
enzyme in such a way that it competes with the normal substrate for 
binding at the active site. A competitive inhibitor reacts reversibly with 
the enzyme to form an enzyme-inhibitor complex EI!, analogous to the 
enzyme-substrate complex: 
EQU E+I==EI 
Following the Michaelis-Menten formalism, we can define the inhibitor 
constant, K.sub.i, as the dissociation constant of the enzyme-inhibitor 
complex: 
##EQU1## 
Thus, in accordance with the above and as used herein, K.sub.i is 
essentially a measurement of affinity between a molecule, and its 
receptor, or in relation to the present invention, between the present 
inventive compounds and the enzyme to be inhibited. It should be noted 
that IC50 is a related term used when defining the concentration or amount 
of a compound which is required to cause a 50% inhibition of the target 
enzyme. 
The term "inhibition", in the context of tumor growth or tumor cell growth, 
may be assessed by delayed appearance of primary or secondary tumors, 
slowed development of primary or secondary tumors, decreased occurrence of 
primary or secondary tumors, slowed or decreased severity of secondary 
effects of disease, arrested tumor growth and regression of tumors, among 
others. In the extreme, complete inhibition, is referred to herein as 
prevention. 
The term "prevention", in relation to tumor growth or tumor cell growth, 
means no tumor or tumor cell growth if none had occurred, no further tumor 
or tumor cell growth if there had already been growth. 
The term "prostate disease" relates to prostate cancer such as 
adenocarcinoma or metastatic cancers, conditions characterized by abnormal 
growth of prostatic epithelial cells such as benign prostatic hyperplasia, 
and other conditions requiring treatment by the compounds of the present 
invention. 
The compounds and compositions of the present invention useful for 
treatment of cancer, include but are not limited to types of cancer 
selected from the following group: ACTH-producing tumors, acute 
lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal 
cortex, bladder cancer, brain cancer, breast cancer, cervix cancer, 
chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal 
cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, 
Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head & neck 
cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, 
lung cancer(small and/or non-small cell), malignant peritoneal effusion, 
malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, 
neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary 
(germ cell) cancer, pancreatic cancer, penis cancer, prostate cancer, 
retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell 
carcinomas, stomach cancer, testicular cancer, thyroid cancer, 
trophoblastic neoplasms, cancer of the uterus, vaginal cancer, cancer of 
the vulva, and Wilm's tumor. 
The term "ischemia" relates to localized tissue anemia due to obstruction 
of the inflow of arterial blood. Global ischemia occurs under conditions 
in which blood flow to the entire brain ceases for a period of time, such 
as may result from cardiac arrest. Focal ischemia occurs under conditions 
in which a portion of the brain is deprived of its normal blood supply, 
such as may result from thromboembolytic occlusion of a cerebral vessel, 
traumatic head injury, edema, and brain tumors. 
The term "nervous tissue" refers to the various components that make up the 
nervous system including neurons, neural support cells, glia, Schwann 
cells, vasculature contained within and supplying these structures, the 
central nervous system, the brain, the brain stem, the spinal cord, the 
junction of the central nervous system with the peripheral nervous system, 
the peripheral nervous system and allied structures. 
The term "nervous function" refers to the various functions of the nervous 
system and its parts which are manifest in sensing the environment, 
awareness of it, homeostasis to it and interaction with it as shown, by 
example, in the ability to perform activities of daily living, work, 
cogitation and speech. 
The term "nervous insult" refers to damage to nervous tissue which includes 
brain and nervous tissue damage and destruction, in whole or in part, and 
resultant morbidity, disability, neurologic deficia and death. Nervous 
insult can be from various origins including ischemia, hypoxia, 
cerebrovascular accident, metabolic, toxic, neurotoxic, trauma, surgery, 
iatrogenic, pressure, mass effect, hemorrhage, thermal, chemical, 
radiation, vasospasm, neurodegenerative disease, neurodegenerative 
process, infection, Parkinson's disease, amyotrophic lateral sclerosis, 
myelination/demyelination processes, epilepsy, cognitive disorders, 
glutamate abnormalities, and their secondary effects. 
The term "glutamate abnormalities" refers to any condition, disease, or 
disorder that involves glutamate, and includes but is not limited to the 
nervous insults listed above. 
The term "glutamate modulator" refers to any composition of matter, alone 
or in combination with another agent, which affects the level of glutamate 
in an animal, including a human being. 
The term "neuroprotective" is an effect which reduces, arrests, or 
ameliorates nervous insult and is protective, resuscitative or revivative 
for nervous tissue that has suffered nervous insult. 
The term "treatment" refers to any process, action, application, therapy, 
or the like, wherein an animal, including a human being, is subject to 
medical aid with the object of improving the animal's condition, directly 
or indirectly. 
The term "Compound 3" refers to the compound 
2-(Phosphonomethyl)pentanedioic Acid. 
Synthesis of NAALADase Inhibitors 
It has been unexpectedly found that compounds with the following general 
structure were found to be very potent inhibitors of NAALADase: 
##STR5## 
These compounds may be prepared by the general method of Ji et al., 
Synthesis, 1988, 444-448. Their synthesis is outlined in Schemes 1, 2, and 
3 below. 
All of the above-described inhibitors can be synthesized by standard 
organic synthetic procedures. The precursor compounds of the present 
invention can be easily made by a ordinary person skill in the art 
utilizing known methods, such as Scheme 1 below. Production of compounds 
containing the R group substitutions can be easily made utilizing known 
methods. See, for example, Froestl et al., J. Med. Chem., 1995, 38, 
3313-3331, Phosphinic Acid Analogues of GABA. 
##STR6## 
Further methods of synthesizing phosphinic acid esters are also described 
in J. Med. Chem., 1988, 31, 204-212, and may be found in Scheme II, below. 
##STR7## 
Starting with the aforementioned phosphinic acid esters, there are a 
variety of routes that can be used to prepare the compounds of the present 
invention. For example, a general route was recently described in Ji et 
al., Synthesis, 1988, 444-448, and is set forth below in Scheme III. 
##STR8## 
In Vitro Inhibition of NAALADase Activity 
Three compounds were tested for inhibition of NAALADase activity: 
2-(phosphonomethyl) pentanedioic acid, 2-(phosphonomethyl)succinic acid, 
and 2-2-carboxyethyl)hydroxyphosphinol!methyl!-pentanedoic acid. The 
results are shown in Table II. 
TABLE II 
______________________________________ 
in vitro Activity of NAALADase Inhibitors 
compd K.sub.i (nM) 
______________________________________ 
2-(phosphonomethyl)pentanedioic acid 
0.275 .+-. 0.08 
2-(phosphonomethyl)succinic acid 
700. .+-. 67.3 
2-2-carboxyethyl)hydroxyphosphinol! 
1.89 .+-. 0.19 
methyl!-pentanedoic acid) 
______________________________________ 
2-(phosphonomethyl)pentanedioic acid showed a high level of NAALADase 
inhibiting activity, with a K.sub.i of 0.27 nM (Table II). The activity of 
this compound is &gt;1000 times more potent than that of previously described 
inhibitors. The procedure for assaying the compounds is set forth below. 
NAALADase activity was assayed as described. In brief, the assay measured 
the amount of .sup.3 H!Glu liberated from .sup.3 H!NAAG in 50 mM Tris-Cl 
buffer in 15 min at 37.degree. C. using 30-50 .mu.g of synaptosomal 
protein; substrate and product were resolved by anion-exchange liquid 
chromatography. Duplicate assays were always performed so that no more 
than 20% of the NAAG was digested, representing the linear range of 
peptidase activity. Quisqualate (100 .mu.M) was included in parallel assay 
tubes to confirm the specificity of measurements. 
The 2-(phosphonomethyl)succinic acid showed a large decrease in efficacy in 
inhibiting the activity of NAALADase (Table II), suggesting that a 
glutamate analog attached to the phosphonic acid is required for potent 
inhibition of the enzyme. In addition, 2-2-carboxyethyl) 
hydroxyphosphinol!methyl!-pentanedoic acid, which has an additional 
carboxylic acid side chain similar to the aspartate residue found in NAAG, 
did not lead to an increase in potency. 
TOXICOLOGICAL EFFECTS 
The compounds of the present invention have demonstrated that they are 
non-toxic when administered to rats and mice during in vivo neurological 
experiments and would accordingly, not be expected to demonstrate toxic 
effects in humans when administered in therapeutic doses. Furthermore, 
NAALADase inhibitors have not demonstrated toxic side effects upon 
exposure to cell lines. 
In order to explore the potential toxicological effects of NAALADase 
inhibition, a group of mice were injected with a single peritoneal dose of 
2-(phosphonomethyl)pentanedioic acid, a NAALADase inhibitor having a high 
activity. The dosages were given in increasing concentrations of 
milligrams (mg) per kilogram (kg) of body weight. Dosages of 1, 5, 10, 30, 
100, 300, and 500 mg/kg (of body weight) were administered and no acute 
adverse effects were observed at any dose tested. The mice were 
subsequently observed two times per day for 5 consecutive days. Table III 
gives the percent survival rate for the doses tested. 
TABLE III 
______________________________________ 
NAALADase Inhibitor 
DOSES OF COMPOUND 
mg/kg 1 5 10 30 100 300 500 
______________________________________ 
% of animal 
100 100 100 100 100 100 66.7 
survival as 
of Day 5 
______________________________________ 
PHARMACEUTICALLY ACCEPTABLE DERIVATIVES 
The compounds of the present invention can be used in the form of salts 
derived from inorganic or organic acids and bases. Included among but not 
limited to such acid salts are the following: acetate, adipate, alginate, 
aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, 
camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, 
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, 
glycerophosphate, hemissulfate heptanoate, hexanoate, hydrochloride, 
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, 
methanesulfonate, 2-naphthalensulfonate, nicotinate, oxalate, pamoate, 
pectinate, propionate, succinate, tartrate, thiocyanate, tosylate and 
undecanoate. Included among but not limited to such base salts are the 
following: ammonium salts, alkali metal salts such as sodium and potassium 
salts, alkaline earth metal salts such as calcium and magnesium salts, 
salt with organic bases such as dicyclohexylamine salts, 
N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, 
and so forth. Also, the basic nitrogen-containing groups can be 
quarternized with such agents as lower alkyl halides, such as methyl, 
ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates 
like dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides 
such as decyl, lauryl, myristyl and stearyl chlorides, bromides and 
iodides, aralkyl halides like benzyl and phenethyl bromides and others. 
Water or oil-soluble or dispersible products are thereby obtained. 
ROUTE OF ADMINISTRATION 
For these purposes, the compounds of the present invention may be 
administered orally, parenterally, by inhalation spray, topically, 
rectally, nasally, buccally, vaginally or via an implanted reservoir in 
dosage formulations containing conventional non-toxic 
pharmaceutically-acceptable carriers, adjuvants and vehicles. The term 
parenteral as used herein includes subcutaneous, intravenous, 
intramuscular, intraperitoneally, intrathecally, intraventricularly, 
intrasternal and intracranial injection or infusion techniques. Generally, 
at the present time, invasive techniques are preferred, particularly 
administration directly to damaged neuronal tissue. 
In addition, administration may be by a single dose, it may be repeated at 
intervals or it may be by continuous infusion. Where continuous infusion 
is preferred, pump means often will be particularly preferred for 
administration. Especially, subcutaneous pump means may be preferred in 
this regards. 
Since NAALADase inhibitors are small, easily diffusible, and relatively 
stable, it is well suited to long-term continuous administration, such as 
by a perfusion pump. Also, it may be desirable to administer NAALADase 
inhibitors and other agents of the present invention by intraventricular 
injection to the affected CNS tissue on a regular basis. 
Compositions and methods of the invention also may utilize controlled 
release technology. Thus, for example, NAALADase inhibitors may be 
incorporated into a hydrophobic polymer matrix for controlled release over 
a period of days. Such controlled release films are well known to the art. 
Examples of polymers commonly employed for this purpose that may be used 
in the present invention include nondegradable ethylene-vinyl acetate 
copolymer degradable lactic acid-glycolic acid copolymers, and liposomal 
polymers. Certain hydrogels such as poly(hydroxyethylmethacrylate) or 
poly(vinylalcohol) also may be useful, but for shorter release cycles then 
the other polymer releases systems, such as those mentioned above. 
To be effective therapeutically and avoid unwanted neurological effects 
which may or may not be caused by NAALADase inhibitors in neural tissue, 
the composition should be formulated such that it will not readily 
penetrate the blood-brain barrier in significant amounts when peripherally 
administered. However, for compositions which are administered locally, 
such by intraperitoneal injection or by polymeric implant, such 
neurological concerns may be obviated. 
The pharmaceutical compositions may be in the form of a sterile injectable 
preparation, for example as a sterile injectable aqueous or oleaginous 
suspension. This suspension may be formulated according to techniques know 
in the art using suitable dispersing or wetting agents and suspending 
agents. The sterile injectable preparation may also be a sterile 
injectable solution or suspension in a non-toxic parenterally-acceptable 
diluent or solvent, for example as a solution in 1,3-butanediol. Among the 
acceptable vehicles and solvents that may be employed are water, Ringer's 
solution and isotonic sodium chloride solution. In addition, sterile, 
fixed oils are conventionally employed as a solvent or suspending medium. 
For this purpose any bland fixed oil may be employed including synthetic 
mono- or diglycerides. Fatty acids such as oleic acid and its glyceride 
derivatives find use in the preparation of injectables, olive oil or 
castor oil, especially in their polyoxyethylated versions. These oil 
solutions or suspensions may also contain a long-chain alcohol diluent or 
dispersant. 
The compounds may be administered orally in the form of capsules or 
tablets, for example, or as an aqueous suspension or solution. In the case 
of tablets for oral use, carriers which are commonly used include lactose 
and corn starch. Lubricating agents, such as magnesium stearate, are also 
typically added. For oral administration in a capsule form, useful 
diluents include lactose and dried corn starch. When aqueous suspensions 
are required for oral use, the active ingredient is combined with 
emulsifying and suspending agents. If desired, certain sweetening and/or 
flavoring and/or coloring agents may be added. 
The compounds of this invention may also be administered in the form of 
suppositories for rectal administration of the drug. These compositions 
can be prepared by mixing the drug with a suitable non-irritating 
excipient which is solid at room temperature but liquid at rectal 
temperature and therefore will melt in the rectum to release the drug. 
Such materials include cocoa butter, beeswax and polyethylene glycols. 
The compounds of this invention may also be administered optically, 
especially when the conditions addressed for treatment involve areas or 
organs readily accessible by topical application, including neurological 
disorders of the eye, the skin, or the lower intestinal tract. Suitable 
topical formulations are readily prepared for each of these areas. For 
ophthalmic use, the compounds can be formulated as micronized suspensions 
in isotonic, pH adjusted sterile saline, or, preferably, as solutions is 
isotonic, pH adjusted sterile saline, either with or without a 
preservative such as benzylalkonium chloride. Alternatively for the 
ophthalmic uses the compounds may be formulated in an ointment such as 
petrolatum. 
For application topically to the skin, the compounds can be formulated in a 
suitable ointment containing the compound suspended or dissolved in, for 
example, a mixture with one or more of the following: mineral oil, liquid 
petrolatum, white petrolatum, propylene glycol, polyoxyethylene 
polyoxypropylene compound, emulsifying wax and water. Alternatively, the 
compounds can be formulated in a suitable lotion or cream containing the 
active compound suspended or dissolved in, for example, a mixture of one 
or more of the following: mineral oil, sorbitan monostearate, polysorbate 
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol 
and water. 
Topical application for the lower intestinal tract may be effected in a 
rectal suppository formulation (see above) or in a suitable enema 
formulation. 
DOSE 
Dosage levels on the order of about 0.1 mg to about 10,000 mg of the active 
ingredient compound are useful in the treatment of the above conditions, 
with preferred levels of about 0.1 mg to about 1,000 mg. The amount of 
active ingredient that may be combined with the carrier materials to 
produce a single dosage form will vary depending upon the host treated and 
the particular mode of administration. 
It is understood, however, that a specific dose level for any particular 
patient will depend upon a variety of factors including the activity of 
the specific compound employed, the age, body weight, general health, sex, 
diet, time of administration, rate of excretion, drug combination, and the 
severity of the particular disease being treated and form of 
administration. 
Treatment dosages generally may be titrated to optimize safety and 
efficacy. Typically, dosage-effect relationships from in vitro initially 
can provide useful guidance on the proper doses for patient 
administration. Studies in animal models also generally may be used for 
guidance regarding effective dosages for treatment of cancers in 
accordance with the present invention. In terms of treatment protocols, it 
should be appreciated that the dosage to be administered will depend on 
several factors, including the particular analog that is administered, the 
route administered, the condition of the particular patient, etc. In that 
most of these agents have peptidyl portions it will generally be desirable 
to administer the agents I.V., but administration by other routes is 
contemplated where appropriate. Generally speaking, one will desire to 
administer an amount of the agent that is effective to achieve a serum 
level commensurate with the concentrations found to be effective in vitro. 
Thus, where an agent is found to demonstrate in vitro activity at, e.g., 
10 .mu.M, one will desire to administer an amount of the drug that is 
effective to provide about a 10 .mu.M concentration in viva. Determination 
of these parameters are well within the skill of the art. 
These considerations, as well as effective formulations and administration 
procedures are well known in the art and are described in standard 
textbooks. 
A particular formulation of the invention uses a lyophilized form of 
NAALADase inhibitor, in accordance with well known techniques. For 
instance, 1 to 100 mg of NAALADase inhibitor may be lyophilized in 
individual vials, together with carrier and buffer compound, for instance, 
such as mannitol and sodium phosphate. The NAALADase inhibitor may be 
reconstituted in the vials with bacteriostatic water and then 
administered, as described elsewhere herein. 
Compositions of the present invention for treating global ischemia are 
administered internally to a subject and contain an effective amount of a 
NAALADase inhibitor. Doses included in the pharmaceutical compositions are 
of an efficacious, nontoxic quantity. Persons skilled in the art using 
routine clinical testing are able to determine optimum doses. The desired 
dose is administered to a subject from 1 to 6 or more times daily, orally, 
rectally, parenterally, or topically and may follow a higher initial 
amount administered as a bolus dose. 
ADMINISTRATION REGIMEN 
Any effective treatment regimen can be utilized and readily determined and 
repeated as necessary to effect treatment. 
In clinical practice, the compositions containing NAALADase inhibitor alone 
or in combination with other therapeutic agents are administered in 
specific cycles until a response is obtained. 
a. Administration for Nervous Insult 
The present invention is directed to regimens which dictate the timing and 
sequence of delivery of treatment medications to include pretreatment. To 
maximize protection of nervous tissue from nervous insult, the compounds 
of the present invention should be administered is as soon as possible 
within the affected cells. This would include administration before the 
nervous ischemic insult in situations of increased likelihood of ischemia 
or stroke. Known in anticipatory situations of include surgery (cartoid 
endarterectomy, cardiac, vascular, aortic, orthopedic), endovascular 
procedures such as any type of arterial catherization (cartoid, vertebral, 
aortic, cardia, renal, spinal, Adamkiewicz and others) for diagnostic or 
therapeutic purposes including evaluation and treatment of vascular 
stenosis, aneurysm or arteriovenous malformation and or injection of 
embolic agents, coils or balloons for hemostasis, interruption of 
vascularity or treatment of brain lesions, predisposing medical 
conditions, including crescendo transient ischemic attacks, anticipated 
emboli or sequential strokes. Where pretreatment for stroke or ischemia is 
not possible or practicable, it is important to get the compounds of the 
present invention to the affected cells as quickly as possible during or 
after the event. The time between the stroke, diagnosis and treatment 
should be reduced to its minimum to save the ischemic cells from damage 
and death. 
b. Administration for Prostate Disease/Cancer 
For patients who initially present without advanced or metastatic cancer, 
NAALADase inhibitor based drugs are used as an immediate initial therapy 
prior to surgery and radiation therapy, and as a continuous post-treatment 
therapy in patients at risk for recurrence or metastasis (based upon high 
PSA, high Gleason's score, locally extensive disease, and/or pathological 
evidence of tumor invasion in the surgical specimen). The goal in these 
patients is to inhibit the growth of potentially metastatic cells from the 
primary tumor during surgery or radiotherapy and inhibit the growth of 
tumor cells from undetectable residual primary tumor. 
For patients who initially present with advanced or metastatic cancer, 
NAALADase inhibitor based drugs are used as a continuous supplement to, or 
possible as a replacement for hormonal ablation. The goal in these 
patients is to slow tumor cell growth from both the untreated primary 
tumor and from the existing metastatic lesions. 
In addition, the invention may be particularly efficacious during 
post-surgical recovery, where the present compositions and methods may be 
particularly effective in lessening the chances of recurrence of a tumor 
engendered by shed cells that cannot be removed by surgical intervention. 
COMBINATION WITH OTHER TREATMENTS 
a. Nervous Insult 
In compositions for treating stroke, particularly acute ischemic stroke, 
and global ischemia caused by drowning, head trauma and so forth, the 
NAALADase inhibitor can be co-administered with one or more agents active 
in reducing the risk of stroke, such as aspirin or ticlopidine (preferably 
ticlopidine, which has been demonstrated to reduce the risk of a second 
ischemic event). Co-administration can be in the form of a single 
formulation (combining, for example, a NAALADase inhibitor and ticlopidine 
with pharmaceutically acceptable excipients, optionally segregating the 
two active ingredients in different excipient mixtures designed to 
independently control their respective release rates and durations) or by 
independent administration of separate formulations containing the active 
agents. 
If desired, the pharmaceutical composition to be administered may also 
contain minor amounts of non-toxic auxiliary substances such as wetting or 
emulsifying agents, pH buffering agents and the like, such as for example, 
sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. 
The compounds of this invention are generally administered in a 
pharmaceutical composition which comprises a pharmaceutical excipient or 
excipients in combination with a NAALADase inhibitor. The level of the 
drug in a formulation can vary within the full range employed by those 
skilled in the art, namely, from about 0.01 percent weight (% w) to about 
99.99% w of the drug based on the total formulation and about 0.01% w to 
99.99% w excipient. Preferably, the formulation will be about 3.5 to 60% 
by weight of the NAALADase inhibitor, with the rest being suitable 
pharmaceutically excipients. 
b. Prostate Disease/Cancer 
Surgery and Radiation 
In general, surgery and radiation therapy are employed as potentially 
curative therapies for patients under 70 years of age who present with 
clinically localized disease and are expected to live at least 10 years. 
Approximately 70% of newly diagnosed prostate cancer patients fall into 
this category. Approximately 90% of these patients (65% of total patients) 
undergo surgery, while approximately 10% of these patients (7% of total 
patients) undergo radiation therapy. 
Histopathological examination of surgical specimens reveals that 
approximately 63% of patients undergoing surgery (40% of total patients) 
have locally extensive tumors or regional (lymph node)metastasis that was 
undetected at initial diagnosis. These patients are at a significantly 
greater risk of recurrence. Approximately 40% of these patients will 
actually develop recurrence within five years after surgery. Results after 
radiation are even less encouraging. Approximately 80% of patients who 
have undergone radiation as their primary therapy have disease persistence 
or develop recurrence or metastasis within five years after treatment. 
Currently, most of these surgical and radiotherapy patients generally do 
not receive any immediate follow-up therapy. Rather, they are monitored 
frequently for elevated Prostate Specific Antigen ("PSA"), which is the 
primary indicator of recurrence or metastasis. 
Thus, there is considerable opportunity to use the present invention in 
conjunction with surgical intervention. 
Hormonal Therapy 
Hormonal ablation is the most effective palliative treatment for the 10% of 
patients presenting with metastatic prostate cancer at initial diagnosis. 
Hormonal ablation by medication and/or orchiectomy is used to block 
hormones that support the further growth and metastasis of prostate 
cancer. With time, both the primary and metastatic tumors of virtually all 
of these patients become hormone-independent and resistant to therapy. 
Approximately 50% of patients presenting with metastatic disease die 
within three years after initial diagnosis, and 75% of such patients die 
within five years after diagnosis. Continuous supplementation with 
NAALADase inhibitor based drugs are used to prevent or reverse this 
potentially metastasis-permissive state. 
Chemotherapy 
Chemotherapy has been more successful with some cancers than with others. 
It is likely that the combination of chemotherapy with therapies of the 
present invention in some cases will be synergistic. However, chemotherapy 
currently has little effect on prostate cancer and is generally reserved 
as a last resort, with dismal results. For this type of cancer, the 
opportunity to combine chemotherapy with methods and compositions of the 
invention will be rare. 
Immunotherapy 
The NAALADase inhibitors may also be used in combination with monoclonal 
antibodies in treating prostate cancer. Because pelvic lymph node 
involvement affects the 5-year survival rate--84% of patients without 
pelvic lymph node involvement survive 5 years, compared with only 34% of 
those having pelvic lymph node involvement, the use of NAALADase 
inhibitors in combination with monoclonal antibodies becomes significant. 
A specific example of such an antibody includes cell membrane-specific 
anti-prostate antibody. 
The present invention may also be used with immunotherapies based on 
polyclonal or monoclonal antibody-derived reagents, for instance. 
Monoclonal antibody-based reagents are most preferred in this regard. Such 
reagents are well known to persons of ordinary skill in the art. 
Radiolabelled monoclonal antibodies for cancer therapy, such as the 
recently approved use of monoclonal antibody conjugated with strontium-89, 
also are well known to persons of ordinary skill in the art. 
Cryotherapy 
Cryotherapy recently has been applied to the treatment of some cancers. 
Methods and compositions of the present invention also could be used in 
conjunction with an effective therapy of this type. 
Combinations with other Active Agents 
According to another aspect of the invention, pharmaceutical compositions 
of matter useful for inhibiting cancer are provided that contain, in 
addition to the aforementioned compounds, an additional therapeutic agent. 
Such agents may be chemotherapeutic agents, ablation or other therapeutic 
hormones, antineoplastic agents, monoclonal antibodies useful against 
cancers and angiogenesis inhibitors. The following discussion highlights 
some agents in this respect, which are illustrative, not limitative. A 
wide variety of other effective agents also may be used. 
Among hormones which may be used in combination with the present inventive 
compounds, diethylstilbestrol (DES), leuprolide, flutamide, cyproterone 
acetate, ketoconazole and amino glutethimide are preferred. 
Among antineoplastic and anticancer agents that may be used in combination 
with the inventive compounds, 5-fluorouracil, vinblastine sulfate, 
estramustine phosphate, suramin and strontium-89 are preferred. Other 
chemotherapeutics useful in combination and within the scope of the 
present invention are buserelin, chlorotranisene, chromic phosphate, 
cisplatin, cyclophosphamide, dexamethasone, doxorubicin, estradiol, 
estradiol valerate, estrogens conjugated and esterified, estrone, ethinyl 
estradiol, floxuridine, goserelin, hydroxyurea, melphalan, methotrexate, 
mitomycin and prednisone. 
Table IV provides known median dosages for selected cancer agents which may 
be useful in combination with the compounds and compositions of the 
present invention. It should be noted that specific dose levels for the 
chemotherapeutic agents below will depend upon similar dosing 
considerations as those listed in the DOSAGE section for NAALADase 
inhibitors presented herein. 
TABLE IV 
______________________________________ 
NAME OF CHEMOTHERAPEUTIC 
AGENT MEDIAN DOSAGE 
______________________________________ 
Asparaginase 10,000 units 
Bleomycin Sulfate 15 units 
Carboplatin 50-450 mg. 
Carmustine 100 mg. 
Cisplatin 10-50 mg. 
Cladribine 10 mg. 
Cyclophosphamide 100 mg.-2 gm. 
(lyophilized) 
Cyclophosphamide (non- 
100 mg.-2 gm. 
lyophilized) 
Cytarabine (lyophilized 
100 mg.-2 gm. 
powder) 
Dacarbazine 100 mg.-200 mg. 
Dactinomycin 0.5 mg. 
Daunorubicin 20 mg. 
Diethylstilbestrol 250 mg. 
Doxorubicin 10-150 mg. 
Etidronate 300 mg. 
Etoposide 100 mg. 
Floxuridine 500 mg. 
Fludarabine Phosphate 50 mg. 
Fluorouracil 500 mg.-5 gm. 
Goserelin 3.6 mg. 
Granisetron Hydrochloride 
1 mg. 
Idarubicin 5-10 mg. 
Ifosfamide 1-3 gm. 
Leucovorin Calcium 50-350 mg. 
Leuprolide 3.75-7.5 mg. 
Mechlorethamine 10 mg. 
Medroxyprogesterone 1 gm. 
Melphalan 50 gm. 
Methotrexate 20 mg.-1 gm. 
Mitomycin 5-40 mg. 
Mitoxantrone 20-30 mg. 
Ondansetron Hydrochloride 
40 mg. 
Paclitaxel 30 mg. 
Pamidronate Disodium 30-*90 mg. 
Pegaspargase 750 units 
Plicamycin 2,500 mcgm. 
Streptozocin 1 gm. 
Thiotepa 15 mg. 
Teniposide 50 mg. 
Vinblastine 10 mg. 
Vincristine 1-5 mg. 
Aldesleukin 22 million units 
Epoetin Alfa 2,000-10,000 units 
Filgrastim 300-480 mcgm. 
Immune Globulin 500 mg.-10 gm. 
Interferon Alpha-2a 3-36 million units 
Interferon Alpha-2b 3-50 million units 
Levamisole 50 mg. 
Octreotide 1,000-5,000 mcgm. 
Sargramostim 250-500 mcgm. 
______________________________________ 
In Vitro Assay of NAALDase Inhibitors on Ischemia 
Referring now to FIG. 1 of the drawings, the effect of different doses of 
2-(Phosphonomethyl)pentanedioic Acid on the amount of in vitro toxicity 
following ischemic insult in cortical cultures is shown. Concentrations 
ranging from 100 pM to 1 .mu.M of 2-(Phosphonomethyl)pentanedioic Acid 
administered during ischemic insult and for one hour following show a 
sharp decrease in the amount of in vitro toxicity. The percentages 
concerning the toxicity for different doses is shown graphically in FIG. 
1. The numerical percentages are also provided below in Table IV. 
TABLE IV 
______________________________________ 
Dose % Toxicity 
______________________________________ 
Control 100. .+-. 9.0 
(n = 5) 
100 pM 66.57 .+-. 4.38 
(n = 5) 
1 nM 42.31 .+-. 9.34 
(n = 5) 
10 nM 33.08 .+-. 9.62 
(n = 5) 
100 nM 30.23 .+-. 9.43 
(n = 5) 
1 uM 8.56 .+-. 8.22 
(n = 5) 
______________________________________ 
The methods for obtaining the data shown above in Table IV and graphically 
represented in FIG. 1 are set forth in the protocol described in Example 
11, below. 
Ischemia was then induced using potassium cyanide and 2-deoxyglucose (2-DG) 
in a standard technique, such as that described below. 
Cultures and media were then assayed according to standard cytologic cell 
injury assay, such as the LDH Assay set forth and described below. 
In Vitro Toxicity of NAAG on Cortical Cultures 
Referring now to FIG. 2 of the drawings, NAAG toxicity in cortical cell 
cultures is plotted graphically against various dosages of NAAG. Dosages 
of NAAG are administered for 20 minutes and range from 3 uM to 3 mM, and 
include 3 uM, 10 uM, 30 uM, 100 uM, 300 uM, 1 mM, and 3 mM. Numerical 
results of the percentage toxicity are also shown in Table V, below. 
TABLE V 
______________________________________ 
Dose of NAAG % Toxicity 
______________________________________ 
3 .mu.M 3.51 (n = 1) 
10 .mu.M 4.3 .+-. 3.12 
(n = 3) 
30 .mu.M 11.40 .+-. 6.17 
(n = 3) 
100 .mu.M 12.66 .+-. 5.50 
(n = 3) 
300 .mu.M 13.50 .+-. 4.0 
(n = 3) 
1 mM 21.46 .+-. 4.20 
(n = 3) 
3 mM 45.11 .+-. 4.96 
(n = 3) 
______________________________________ 
In Vitro Assay of NAAG toxicity as blocked by Example 3 
Referring now to FIG. 3 of the drawings, NAAG toxicity in cortical cell 
cultures is graphically plotted against NAAG toxicity in the presence of 
2-(Phosphonomethyl)pentanedioic Acid (1 .mu.M). 
2-(Phosphonomethyl)pentanedioic Acid is administered during exposure to 
NAAG and for one hour following NAAG exposure. Numerical results of the 
comparative toxicity are also shown in the percentages of Table VI. 
Clearly, comparing the results of FIG. 2/Table V and FIG. 3/Table VI show 
the remarkable protective effects of the compounds of the present against 
nervous insult or neuronal damage. 
TABLE VI 
______________________________________ 
Dose of NAAG % Toxicity with Example 3 
______________________________________ 
3 .mu.M -4.71 (n = 1) 
10 .mu.M -3.08 .+-. 0.81 
(n = 3) 
30 .mu.M -4.81 .+-. 1.13 
(n = 3) 
100 .mu.M -2.87 .+-. 0.78 
(n = 3) 
300 .mu.M -2.09 .+-. 0.48 
(n = 3) 
1 mM 0.26 .+-. 1.11 
(n = 3) 
3 mM 16.83 .+-. 8.76 
(n = 3) 
______________________________________ 
Since NAAG is cleaved by NAALADase to release glutamate, adding NAAG to 
cortical cultures in the absence of NAALADase inhibitors is shown to be 
toxic in FIG. 2 (control). FIGS. 2 and 3 show that the addition of 
NAALADase inhibitors along with NAAG provides protection against 
glutamate-induced neurotoxicity in vitro. 
NAALADASE Inhibitors are Protective When Administered Post-Ischemia in 
Cortical Cultures 
Referring now to FIG. 4 of the drawings, ischemic toxicity in cortical 
cultures is graphically plotted against the time of administration of 
2-(Phosphonomethyl)pentanedioic Acid. 2-(Phosphonomethyl)pentanedioic Acid 
is administered during the exposure to the ischemic insult and for one 
hour following (exposure and recovery), for one hour post ischemic insult 
only (recovery only), and for one hour beginning 30 minutes post ischemic 
insult (delayed 30 minutes). Remarkable in vitro protective effects are 
shown not only when the compounds of the present invention are 
administered during exposure to the ischemic event and during recovery 
from the ischemic event, but also that significant neuronal protection may 
be achieved when administration of the compositions of the present 
invention are delayed 30 minutes. Numerical results of the percentage 
toxicity are also shown in Table VII. 
TABLE VII 
______________________________________ 
Time of Administration 
relative to Ischemic Event 
% Toxicity 
______________________________________ 
CONTROL 100% 
Exposure & Recovery 2.54% 
Recovery Only 9.03% 
Delayed 30 minutes 31.49% 
______________________________________ 
In Vivo Infarct Volume After Administration 
Because the in vitro results using 2-(Phosphonomethyl)pentanedioic Acid 
were so strikingly protective against injury from ischemic insult, the in 
vivo neuroprotection using 2-(Phosphonomethyl)pentanedioic Acid is also 
examined. 
Referring now to FIG. 5 and to Table VIII below, infarct volume measuring 
injury to the cortex is evaluated in rats following middle cerebral artery 
occlusion (see Example 12). Control animals receive saline, other animals 
receive 10 mg/kg of 2-(Phosphonomethyl)pentanedioic Acid followed by 2 
mg/kg/hr of 2-(Phosphonomethyl)pentanedioic Acid for 1 hour, and still 
others receive 100 mg/kg of 2-(Phosphonomethyl)pentanedioic Acid followed 
by 20 mg/kg/hr of 2-(Phosphonomethyl)pentanedioic Acid for one hour. 
Again, in vivo protective effects, as demonstrated by the significantly 
reduced injury volume are shown when the compounds of the present 
invention are administered during exposure to the ischemic event. Results 
of the infarct volume testing, shown graphically in FIG. 5 and numerically 
below in Table VIII, show that in high dose administration of compounds of 
the present invention significant protection of the cortex may be achieved 
in vivo. 
TABLE VIII 
______________________________________ 
Cortical Injury Volume (mm3) .+-. S.E.M. 
Vehicle 184.62 .+-. 33.52 (n = 10) 
10 mg/kg Example 3 
135 .+-. 32.18 (n = 10) 
100 mg/kg Example 3 
65.23 .+-. 32.18 (n = 10) 
Cortical Injury Volume (% injury) .+-. S.E.M. 
vehicle 34.44 .+-. 6.53 (n = 10) 
10 mg/kg Example 3 
29.14 .+-. 7.68 (n = 10) 
100 mg/kg Example 3 
13.98 .+-. 6.64 (n = 10) 
Cortical Protection 
Vehicle 0% 
10 mg/kg Example 3: 
27% 
100 mg/kg Example 3 
65% 
______________________________________ 
In Vitro Neurotoxicity Assay 
a. Cell Culture 
Dissociated cortical cultures are prepared using the papain-dissociation 
method of Heuttner and Baughman (1986) as modified by Murphy and Baraban 
(1990). See Table IX for the Dissociated Culture Protocol as used herein. 
Fetuses of embryonic day 17 are removed from timed pregnancy rats (Harlan 
Sprague Dawley). The cortex is rapidly dissected out in Dulbecco's 
phosphate-buffered saline, stripped of meninges, and incubated in a papain 
solution for 15 min at 37.degree. C. The tissue is then mechanically 
triturated and pelleted at 500 g (1000-2000 rpm on swinging bucket 
Beckman). The pellet is resuspended in a DNAase solution, triturated with 
a 10 mL pipette x15-20, layered over a "10.times.10" solution containing 
albumin and trypsin inhibitor (see Table IX for an example of a 
"10.times.10" solution), repelleted, and resuspended in a plating medium 
containing 10% fetal bovine serum (HyClone A-1111-L), 5% heat-inactivated 
Equine serum (HyClone A-3311-L), and 84% modified Earle's basal medium 
(MEM)(Gibco 51200-020) with high glucose (4.5 g/L), and 1 g/L NaHCO.sub.3. 
Each 24-well plate is pretreated with poly-D-lysine (0.5 ml/well of 10 
.mu.g/ml) for 1 h and rinsed with water before plating. Cultures are 
plated at 2.5.times.10.sup.6 cells/ml with each well of a 24 well plate 
receiving 500 .mu.l/well. Alternatively, 35 mm dishes can be plated at 2 
mls/dish, 6 well plates at 2 mls/well, or 12 well plates at 1 ml/well. 
After plating, 50% of the medium is changed every 3-4 days with growth 
serum containing 5% heat-inactivated Equine serum (HyClone A-3311-L), 95% 
modified Earle's basal medium (MEM)(Gibco 51200-020), and 1% L-Glutamine 
(Gibco 25030-081). Experiments are performed after 21 days in cultures. 
Cultures are maintained in a 5% CO.sub.2 atmosphere at 37.degree. C. A 
detailed description of these methodologies is further described in the 
table below. 
TABLE IX 
______________________________________ 
DISSOCIATED CULTURE PROTOCOL 
______________________________________ 
I. PREE SOLUTIONS 
______________________________________ 
Stocks/Solutions: 
DNAase Stock, 1 mL 
Dulbecco's PBS, 500 mL 
(100.times.) 4 gms NaCl (J. T. Baker 
5 mgs of DNAase I 
3624-01); 
(Worthington LS002004); 
1.06 gms Na.sub.2 HPO.sub.4.7H.sub.2) (Fisher 
1 ml dissoc. EBSS 
S373-3); 
Freeze as 50 ul 100 mg KCl (Fisher P217- 
aliquots. 500); 
100 mg KH.sub.2 PO.sub.4 (Sigma P- 
0662); 
500 mls dH.sub.2 O; 
Adjust pH to 7.4 and 
sterile filter. 
Dissociated EBSS, 500 mL 
EDTA Stock, 10 mL 
1.1 gms NaHCO.sub.3 ; 
184.2 mgs EDTA sodium salt 
50 mls EBSS stock (Gibco 
(Sigma ED4S); 
14050-025); 10 mls dH.sub.2 O; 
450 mls dH.sub.2 O; 
Sterile filter. 
Sterile filter. 
10 and 10 Stock, 10 mL 
Poly-D-Lysine Stock, 5 mL 
100 mg BSA (Sigma A- 
5 mg Poly-D-Lysine, 100- 
4919); 150K (Sigma P-6407); 
100 mg Trypsin Inhibitor 
5 mls sterile water; 
from Egg White (Sigma T- 
Keep frozen. 
2011); 
10 mls dissoc. EBSS; 
Sterile filter. 
Media 
Dissociated growth, 500 
Plating media, 300 mL 
mL 250 mls MEM containing 
500 mls MEM (Gibco 
glucose and sodium 
51200-020) containing 
bicarbonate (2.25 gm 
glucose and NaHCO.sub.3 
glucose and 0.5 gm NaHCO.sub.3 in 
(2.25 gm glucose and 0.5 
500 mls Gibco MEM 51200- 
gm NaHCO.sub.3); 020); 
25 mls heat-inactivated 
30 MLS Fetal Bovine Serum 
Equine Serum (HyClone A- 
(HyClone A-1111-L); 
3311-L); 15 mls heat-inactivated 
5 mls L-Glutamine 
Equine Serum (HyClone A- 
(200 mM, 100.times. stock, 
3311-L); 
Gibco 25030-081); 
3 mls L-Glutamine (200 mM, 
Sterile filter. 100.times. stock, Gibco 25030- 
081); 
1 ml Penicillin- 
Streptomycin stock (Gibco 
15140-015); 
Sterile filter. 
For papain dissociation: 
For DNAase treatment: 
4 mg Cysteine (C-8277); 
DNAase, 5 mL 
25 mls dissoc. EBSS; 
4.5 mls dissoc. EBSS; 
250 .mu.l Papain stock 
500 .mu.l "10 and 10" stock; 
(Worthington LS003126); 
50 .mu.l DNAase stock. 
Place in 37.degree. C. waterbath 
`10 and 10`, 5 mL 
until clear. 4.5 mls of EBSS; 
500 .mu.l `10 and 10` stock 
______________________________________ 
II. COAT DISHES 
Use poly-d-lysine stock at 1:100 dilution to coat 24- 
well plates (0.5 ml/well) or at 1:10 dilution to coat 
35 mm glass cover slips (1.0 ml/coverslip). 
Leave until end of dissection. 
III. DISSECT TISSUE 
Use Harlan Sprague-Dawley timed pregnancy rats, 
ordered to arrive at E-17. 
Decapitate, spray abdomen down with 70% EtOH. 
Remove uterus through midline incision and place in 
sterile dPBS. 
Remove brains from embryos, leaving them in dPBS. 
Brain removal: Penetrate skull and skin with 
fine forceps at lambda. Pull back to open posterior 
fossa. Then move forceps anteriorly to separate 
sagittal suture. Brain can be removed by scooping 
back from olfactory bulbs under the brain. 
Move brains to fresh dPBS; subsequently, dissect away 
from cortex. 
IV. PAPAIN DISSOCIATION 
Transfer cortices equally to two 15 ml tubes 
containing sterile papain solution, maintained at 
37.degree. C. 
Triturate .times.1 with sterile 10 ml pipette. 
Incubate only for 15 minutes at 37.degree. C. 
Spin at 500 G for 5 minutes (1000-2000 RPM on swinging 
bucket Beckman). 
V. DNAase TREATMENT 
Remove supernatant and any DNA gel layer from cell 
pellet (or pick up and remove pellet with pipette). 
Move cell pellet to DNAase solution. 
Triturate with 10 ml pipette, .times.15-20. 
Layer cell suspension over the `10 and 10` solution by 
pipetting it against the side of the tubes. 
Spin again at 500 G for 5 minutes. (Cells with spin 
into "10 and 10" LAYER). 
Wash tube sides with plating media without disturbing 
pellett. 
Pipette off the media wash and repeat the wash. 
VI. PLATE 
Add about 4.5 mls plating media to each pellet for 5 
ml volume. 
Re-suspend with 10 ml pipette. 
Pool cells into a single tube. 
Quickly add 10 .mu.l of the suspended cells to a 
hemocytometer so that they don't settle. 
Count cells per large square, corresponding to 10 
million cells/ml. 
Put re-suspended cells into a larger container so that 
they number 2.5 million cells/ml (Thus if there 
Triturate to homogeneity). 
Finish coating plates: 
Aspirate or dump Lysine; 
Wash .times.1 with sterile water and dump. 
Add plating media, with cells, to the plates as 
follows: 
35 mm dishes 2 mls/dish; 
6 well plate 2 mls/well; 
12 well plate 1 ml/well; 
24 well plate 500 .mu.l/well. 
______________________________________ 
VII. FEED 
Cultures are usually made on Thursdays 
Start feeding twice a week; beginning the following 
Monday, feeding Mondays and Fridays. 
Remove 50% of volume and replace with fresh growth 
media. 
______________________________________ 
b. Ischemic Insult using potassium cyanide and 2-deoxyglucose 
Twenty-one to twenty-four days following the initial cortical cell plating, 
the experiment is performed. The cultures are washed three times in HEPES 
buffered saline solution containing no phosphate. The cultures are then 
exposed to potassium cyanide (KCN) (5mM) and 2-deoxyglucose (2-DG) (10 mM) 
for 20 minutes at 37.degree. C. These concentrations were shown previously 
to induce maximal toxicity (Vornov et al., J.Neurochem, 1995, Vol. 65, No. 
4, pp. 1681-1691). At the end of 24 hours, the cultures are analyzed for 
release of the cytosolic enzyme lactate dehydrogenase (LDH), a standard 
measure of cell lysis. LDH measurements are performed according to the 
method of Koh and Choi (J.Neuroscience Methods, 1987; see example 11). 
c. NAAG Induced Neurotoxicity 
Cultures are assessed microscopically and those with uniform neuronal 
densities are used in the NAAG neurotoxicity trials. 
At the time of the experiment, the cultures are washed once in 
HEPES-buffered saline solution (HBSS; NaCl 143.4 mM, HEPES 5 mM, KCl 5.4 
mM, MgSO.sub.4 1.2 mM, NaH.sub.2 PO.sub.4 1.2 mM, CaCl.sub.2 2.0 MM, 
D-glucose 10 mM) (Vornov et al., 1995) and then exposed to various 
concentrations of NAAG for 20 minutes at 37.degree. C. NAAG concentrations 
range from 3 .mu.M to 3 mM, and include 3 .mu.M, 10 .mu.M, 30 .mu.M, 100 
.mu.M, 300 .mu.M, 1 mM, and 3 mM. At the end of exposure, the cells are 
washed once with HEPES buffered saline solution and then replaced with 
serum free modified Earle's basal medium. The cultures are then returned 
to the CO.sub.2 incubator for 24 hour recovery. 
d. Lactate Dehydrogenase Assay 
Release of the cytosolic enzyme lactate dehydrogenase (LDH), a standard 
measure of cell lysis, is used to quantify injury (Koh and Choi, 1987). 
LDH-activity measurements are normalized to control for variability 
between culture preparations (Koh et al., 1990). Each independent 
experiment contains a control condition in which no NAALADase inhibitors 
are added; a small amount of LDH activity is found in these controls. This 
control measurement is subtracted from each experimental point. These 
values are normalized within each experiment as a percentage of the injury 
caused by NAAG/ischemia. Only main effects of NAALADase inhibitors are 
considered; interactions between dose and condition are not examined 
statistically. 
A measurement of the potency of each compound tested is made by measuring 
the percentage of LDH release into the growth media after exposure to 
NAAG/ischemia plus NAALADase inhibitor or NAAG/ischemia plus saline 
(control). Since high concentrations of glutamate may be toxic to cells in 
certain circumstances, measurement of glutamate toxicity is observed using 
LDH as a standard measurement technique. 
In Vivo Neurotoxicity Assay 
a. Materials and method 
A colony of SHRSP rats is bred at Johns Hopkins School of Medicine from 
three pairs of male and female rats obtained from the National Institutes 
of Health (Laboratory, Sciences Section, Veterinary Resources Program, 
National Center for Research Resources, Bethesda, Md.). All rats are kept 
in a virus-free environment and maintained on regular diet (NIH 31, 
Zeigler Bros, Inc.) with water ad libitum. All groups of rats are allowed 
to eat and drink water until the morning of the experiment. 
Transient occlusion of the middle cerebral artery (MCA) is induced by 
advancing a 4-0 surgical nylon suture into the internal carotid artery 
(ICA) to block the origin of the MCA (Koizumi, 1986; Longa, 1989; Chen, 
1992). Briefly, animals are anesthetized with 4% halothane, and maintained 
with 1.0 to 1.5% halothane in air enriched oxygen using a face mask. 
Rectal temperature is maintained at 37.0.degree..+-.0.5.degree. C. 
throughout the surgical procedure using a heating lamp. The right femoral 
artery is cannulated for measuring blood gases (pH, oxygen tension PO2!, 
carbon dioxide tension PCO2!) before and during ischemia, for monitoring 
blood pressure during the surgery. The right common carotid artery (CCA) 
is exposed through a midline incision; a self-retraining retractor is 
positioned between the digastric and mastoid muscles, and the omohyoid 
muscle is divided. The right external carotid artery (ECA) is dissected 
and ligated. The occipital artery branch of the ECA is then isolated and 
coagulated. Next, the right internal carotid artery (ICA) is isolated 
until the pterygopalatine artery is exposed, and carefully separated from 
the adjacent vagus nerve. The pterygopalatine artery is ligated with 4-0 
silk suture close to its origin. 
After the CCA is ligated with 4-0 silk suture, a 4-0 silk suture to prevent 
bleeding from a puncture site, through which a 2.5 cm length of 4-0 
monofilament nylon suture (Ethilon), its tip rounded by heating near a 
electric cautery, is introduced into the ICA lumen. A 6-0 silk suture is 
tightened around the intraluminal nylon suture at the bifurcation to 
prevent bleeding, and the stretched sutures at the CCA and the ICA are 
released. The nylon suture is then gently advanced as far as 20 mm. 
Anesthesia is terminated after 10 minutes of MCA occlusion in both groups, 
and animals awakened 5 minutes thereafter. After 2 hours of ischemia, 
anesthesia is reanesthetized, and reperfusion is performed by withdrawing 
the intraluminal nylon suture until the distal tip became visible at the 
origin of the ICA. 
Arterial pH and PaCO2, and partial pressure of oxygen (PaO2) are measured 
with a self-calibrating Radiometer electrode system (ABL 3; Copenhagen, 
Denmark). Hemoglobin and arterial oxygen content are measured with a 
hemoximeter (Radiometer, Model OSM3; Copenhagen, Denmark). Blood glucose 
is measured with a glucose analyzer (model 2300A, Yellow Springs 
Instruments, Yellow Springs, Ohio). 
Each group is exposed to 2 hours of right MCA occlusion and 22 hours of 
reperfusion. All variables but the rectal temperature are measured at 
baseline, at 15 minutes and 45 minutes of right MCA occlusion. The rectal 
temperature is measured at baseline, at 0 and 15 min of MCA occlusion, and 
at 0 and 22 hours of reperfusion. 
FIG. 5 clearly shows that the compounds of the present invention when 
administered during ischemia significantly reduces injury to the cortex. 
Thus, significant protection of neurons in vivo may be achieved using the 
compounds of the present invention. 
In Vitro Assay of NAALADase Inhibitors on a Cancer Cell Line 
Referring now to FIGS. 6 and 7 of the drawings, the effect of 7-day 
treatment with quisqualate and 2-(phosphonomethyl)pentanedioic acid on the 
growth of LNCAP cells (a prostate cancer cell line) is shown in FIGS. 6 
and 7, respectively. Concentrations ranging from 10 nM to 1 .mu.M of 
quisqualate and 100 pM to 10 nM of 2-(phosphonomethyl)pentanedioic acid 
show a sharp dose-dependent decrease of LNCAP cell proliferation as 
indicated by the significant decrease in the incorporation of 
3H!thymidine. The data for FIGS. 6 and 7 is shown in Table X, below. 
TABLE X 
______________________________________ 
.sup.3 H-Thymidine Incorporation (DPMs) 
2(phosphonomethyl) 
Dose Quisqualic Acid 
pentanedioic Acid 
______________________________________ 
Control 4813 .+-. 572 
4299 .+-. 887 
10 pM -- 3078 .+-. 1006 
100 pM -- 2062 .+-. 595 
1 nM 3668 .+-. 866 
1001 .+-. 52 
10 nM 2137 .+-. 764 
664 .+-. 366 
100 nM 1543 .+-. 312 
-- 
1 uM 1295 .+-. 181 
-- 
______________________________________ 
The data for Table X is obtained according to the following protocol. Cells 
in RPMI 1640 medium containing 10% Fetal Calf Serum (FCS) are plated in 24 
well plates and allowed to adhere for 24 hours before addition of 
quisqualic acid (10.sup.-9 to 10.sup.-6) or 
2-(phosphonomethyl)pentanedioic acid (10.sup.-11 to 10.sup.-8) for 7 days. 
On the 7th day, the cells are pulsed with 3H-Thymidine for 4 hours, 
harvested and radioactivity measured. Values represent means.+-.SEM of 6 
separate cell wells for each treatment. All experiments are performed at 
least twice. 
To control for the non-specific cytostatic effects of these NAALADase 
inhibitors, the effects of these agents are simultaneously evaluated on 
the non-NAALADase containing prostate cell line, DU145 (Carter et al., 
Proc. Natl. Acad. Sci. USA, (93) 749-753; 1996). The effect of 7-day 
treatment of quisqualate and 2-(phosphonomethyl)pentanedioic acid at 
concentrations up to .mu.M have no significant effect on cell growth. 
These observations provide evidence that the NAALADase inhibition 
properties of these agents are uniquely responsible for their cytostatic 
effects on prostate carcinoma cell lines. 
Cell Lines and Tissue Culture 
LNCAP cells are obtained from Dr. William Nelson at the Johns Hopkins 
School of Medicine in Baltimore, Md. DU145 cells are obtained from 
American Type Culture Collection (Rockville, Md.). Cells are grown in 
RPMI-1640 media supplemented with 5% heat-inactivated fetal calf serum, 2 
mM-glutamine, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin 
(Paragon) in a humidified incubator at 37.degree. C. in a 5% CO.sub.2 /95% 
air atmosphere. 
3H! Thymidine Incorporation Assays 
The cells are suspended at 1.times.10.sup.3 cells/ml in RPMI-1640 media and 
seeded into 24-well plates at 500 .mu.l per well. After 24 hour 
incubation, various concentrations of quisqualic acid (Sigma) or the 
potent NAALADase inhibitor 2-(phosphonomethyl)pentanedioic acid 
(synthesized according to the methods of Jackson et al., J Med Chem 39(2) 
619-622) is added to the wells and the plates are returned to the 
incubator. On days 3, 5, and 7, media and drug are refreshed. On the 8th 
day following seeding, each well is pulsed with 1 .mu.Ci .sup.3 
H-thymidine (New England Nuclear) for 4 hours. Media is then removed and 
the wells washed 2 times with phosphate buffered saline (pH=7.4). The 
contents of each well is subsequently solubilized 250 .mu.l of 0.2N NaOH 
and transferred to scintillation vials. Five mls of UltimaGold (Packard) 
scintillation cocktail is added and radioactivity is quantitated using a 
Beckman LS6001 scintillation counter. 
The purity and/or identity of all synthetic compounds is ascertained by 
thin layer chromatography, High Pressure Liquid Chromatography (HPLC), 
mass spectrometry, and elemental analysis. Proton Nuclear Magnetic 
Resonance (NMR) spectra are obtained using a Bruker spectrometer. Chemical 
shifts are reported in parts per million relative to tetramethylsilane as 
internal standard. Analytical thin-layer chromatography (TLC) is conducted 
on prelayered silica gel GHLF plates (Analtech, Newark, Del.). 
Visualization of the plates is accomplished by using UV light, 
phosphomolybdic acid-ethanol, and/or iodoplatinate charring. Flash 
chromatography is conducted on Kieselgel 60, 230-400 mesh (E. Merck, 
Darmstadt, West Germany). Solvents are either reagent or HPLC grade. 
Reactions are run at ambient temperature and under a nitrogen atmosphere 
unless otherwise noted. Solutions are evaporated under reduced pressure on 
a Buchi rotary evaporator. 
The following examples are illustrative of preferred embodiments of methods 
of preparation of compounds of the invention and are not to be construed 
as limiting the invention thereto. Unless otherwise indicated, all 
percentages are based upon 100% of the final formulations. 
EXAMPLE 1 
This example demonstrates the preparation of Dibenzyl 
2-Methylenepentanedioate using the general method described in Jackson et 
al., J. Med Chem., 1996, 39, 619-622. 
Benzyl acrylate (19.4 g, 120 mmol) was cooled in a two neck 250 ml round 
bottom flask to approximately 5.degree. C. To this was added dropwise HMPT 
(2.14 g, 133.1 mmol) at such a rate as to maintain a temperature of 
5.degree.-10.degree. C. Once addition was complete the ice/water bath was 
removed and the mixture allowed to warm to room temperature. Stirring was 
continued overnight. The clear yellow liquid was added directly to a 
silica gel column (4 cm*40 cm) and eluted with a gradient (19:1-9:1) 
solvent system (hexane/EtOAc). The fractions containing the desired 
material were combined and evaporated to give 1 (10.1 g, 52%) as a clear 
and colorless liquid. TLC R.sub.f 0.26 (9:1, Hex./EtOAc). 
1H-NMR (CDCl3) 7.2-7.3 (m, 10H); 6.15 (s, 1H); 5.55 (s, 1H); 5.12 (s, 2H); 
5.08 (s,2H); 2.58-2.68 (m, 2H); 2.48-2.55 (m, 2H). 
EXAMPLE 2 
This example demonstrates the preparation of Dibenzyl 
2-Bis(benzyloxy)phosphoryl!methyl!pentanedioate using the general method 
described in J. Med Chem., 1996, 39, 619-622. 
Dibenzyl phosphite (9.5 g, 36 mmol) in 350 ml of dichloromethane was cooled 
to 0.degree. C. To this stirring solution was added trimethyl aluminum 
(18.2 ml, 2.0M solution in hexane, 36.4 mmol). After 30 minutes 1 (6.0 g, 
37 mmol) in 90 ml of dichloromethane was added dropwise over 10 minutes. 
The clear and colorless solution was then warmed to room temperature and 
left to stir overnight. The mixture was then quenched by the slow addition 
of 5% HCl. After stirring an additional 1.5 hours the lower organic layer 
was removed and the aqueous layer extracted once with 100 ml of 
dichloromethane. The organics were combined, dried (MgSO.sub.4), and 
evaporated to give a clear light golden liquid. The liquid was 
chromatographed on silica gel (4 cm*30 cm) and eluted with a gradient 
(4:1-1:1) solvent system (Hexane/EtOAc). The fractions containing the 
desired product were combined and evaporated to yield 2 (7.1 g, 42%) as a 
clear and colorless liquid. The liquid was then distilled on a Kughleror 
apparatus at 0.5 mm Hg and 195.degree.-200.degree. C. The distillate was 
discarded and the remaining light golden oil was chromatographed on silica 
gel (1:1, Hex./EtOAc) to give 2.9 g of 2 as a clear and colorless oil. TLC 
R.sub.f 0.5 (1:1, Hex./EtOAc). 
1H-NMR (CDCl.sub.3) 7.1-7.4 (m, 20H); 5.05 (s, 2H); 4.8-5.03 (m, 6H); 2.8 
(1H); 2.22-2.40 (m, 3H); 1.80-2.02 (m, 3H). 
EXAMPLE 3 
This example demonstrates the preparation of 
2-(Phosphonomethyl)pentanedioic Acid (Compound 3) using the general method 
described in J. Med Chem., 1996, 39, 619-622. 
The benzyl pentanedioate 2(2.9 g, 4.9 mmol) was added to a mixture of 20 ml 
of methanol containing 0.29 g (6mol %) of 10% Pd/C. This mixture was 
hydrogenated on a Parr hydrogenator at 40 psi for 24 hours, filtered and 
evaporated to give 3(1.0 g, 90%) as a clear slightly golden viscous oil. 
1H-NMR (D.sub.2 O) 2.6-2.78(m, 1H); 2.25-2.40(m, 2H); 1.75-2.15(m, 4H). 
EXAMPLE 4 
##STR9## 
Benzyl magnesium chloride (750 ml of a 1.0M Et.sub.2 O solution, 0.75 mol) 
was added dropwise to a cooled (0.degree. C.) stirring solution of diethyl 
chlorophosphite (110 g, 0.70 mol) in 750 ml of dry ether. Addition was 
complete after 1.5 hours and the white slurry was warmed to room 
temperature and left to stir for 16 hours. The mixture was then filtered 
and the filtrate evaporated under reduced pressure. Water (140 ml) was 
added followed by a dropwise addition of HCl (10 ml) during which an 
exothermic was observed. After 30 minutes at room temperature, the 
solution was extracted with ethyl acetate. The organics were combined and 
washed with brine, dried (MgSO.sub.4), and evaporated to give a clear 
light yellow liquid. The liquid was then brought up in 80 ml of 10% NaOH 
and stirred for 1 hour. After this time the mixture was washed with ether 
and the aqueous layer acidified to pH 1 with concentrated HCl. The 
solution was then extracted with ethyl acetate. The organics were 
combined, dried (MgSO.sub.4) and evaporated to give 50 g (46%) of a light 
yellow oil. 
EXAMPLE 5 
##STR10## 
Benzylphosphonous acid (6 g, 38.4 mmol) was added to acetonitrile (25 ml) 
and cooled to 10.degree. C. Triethylamine (5.4 ml, 38.7 mmol) was added 
and the temperature maintained at 10.degree. C. Pivaloyl chloride (23.6 
ml, 192 mmol) in 50 ml of 1:1 MeCN/Pyridine was added dropwise over 15 
minutes. Once addition was complete benzyl alcohol (20 ml, 193 mmol) was 
added dropwise over 15 minutes. The mixture was warmed to room temperature 
and stirred for 1 hour. At this time the mixture was acidified with 5% HCl 
and extracted with dichloromethane. The organics were combined, dried 
(MgSO.sub.4), and evaporated to give a clear liquid. The liquid was 
purified by flash chromatography and eluted using a 1:1 hexane/EtOAc 
solvent system. The desired fractions were combined and evaporated under 
reduced pressure to give 4.5 g of a clear and colorless liquid. 
EXAMPLE 6 
##STR11## 
O-Benzyl-benzylphosphonous acid (2 g, 8.1 mmol) was added to carbon 
tetrachloride (20 ml). Triethylamine (4 ml, 29 mmol) was added dropwise 
followed by tosyl dibenzyl glutamate (4 g, 8.0 mmol) in 10 ml of carbon 
tetrachloride. The mixture was stirred for 16 hours and then acidified 
with 5% HCl. The mixture was then extracted with dichloromethane. The 
organics were combined, dried (MgSO.sub.4), and evaporated to give a clear 
light yellow liquid. This was purified by flash chromatography to give a 
clear colorless liquid. The liquid was then hydrogenated at 40 psi in 
water containing 10% Pd/C. Once hydrogenation was complete (24 hours) the 
mixture was filtered through Celite and lyophilized to afford 
N-(Phenylmethyl)hydroxyphosphinyl!glutamic Acid. 
EXAMPLE 7 
A patient is at risk of injury from an ischemic event. The patient would 
then be pretreated with an effective amount of the compounds of the 
present invention, such as set forth in example 6. It would be expected 
that after the pretreatment the patient would be protected from the 
injury. 
EXAMPLE 8 
A patient is suffering from an ischemic event. The patient may then be 
administered, during the event or within a 30 minute window after such an 
event, an effective amount of the compounds of the present invention, such 
as set forth in example 6. It would be expected that the patient would 
recover or would not suffer significant injury due to the ischemic event. 
EXAMPLE 9 
A patient has suffered from an ischemic injury. The patient may then be 
administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that the 
patient would recover from the ischemic injury. 
EXAMPLE 10 
A patient is suffering from a disease characterized by glutamate 
abnormality. The patient may then be administered an effective amount of 
the compounds of the present invention, such as set forth in example 6. It 
would be expected that the patient would be protected from further injury 
caused by the glutamate abnormality or would recover from the disease. 
EXAMPLE 11 
A patient is diagnosed as requiring treatment for glutamate regulation. The 
patient may then be administered an effective amount of the compounds of 
the present invention, such as set forth in example 6. It would be 
expected that the patient's prognosis would improve, the patient would be 
protected from injury associated with glutamate regulation or the patient 
would recover from the disease requiring the treatment. 
EXAMPLE 12 
A patient is suffering from or has suffered a nervous insult, such as that 
arising from a neurodegenerative disease or neurodegenerative process. The 
patient may then be administered an effective amount of the compounds of 
the present invention, such as set forth in example 6. It would be 
expected that the patient would be protected from further injury or would 
recover from the nervous insult. 
EXAMPLE 13 
A patient is suffering from Parkinson's disease. The patient may then be 
administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that the 
patient would be protected from further neurodegeneration or would recover 
from the disease. 
EXAMPLE 14 
A patient is suffering from amyotrophic lateral sclerosis. The patient may 
then be administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that the 
patient would be protected from further neurodegeneration or would recover 
from the disease. 
EXAMPLE 15 
A patient is suffering from epilepsy. The patient may then be administered 
an effective amount of the compounds of the present invention, such as set 
forth in example 6. It would be expected that the patient would be 
protected from further neurodegeneration or would recover from the 
disease. 
EXAMPLE 16 
A patient is suffering from abnormalities in myelination/demyelination 
processes. The patient may then be administered an effective amount of the 
compounds of the present invention, such as set forth in example 6. It 
would be expected that the patient would be protected from further 
neurodegeneration or would recover from the disease. 
EXAMPLE 17 
A patient is diagnosed as suffering from a cerebrovascular accident, such 
as stroke. The patient may then be administered an effective amount of the 
compounds of the present invention, such as set forth in example 6. It 
would be expected that after the treatment the patient would be 
significantly protected from or would recover from injury due to the 
cerebrovascular accident. 
EXAMPLE 18 
A patient is diagnosed as suffering from a head trauma. The patient may 
then be administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that after 
the treatment the patient would be significantly protected from or would 
recover from injury due to an ischemic brain, spinal, or peripheral injury 
resulting from the head trauma. 
EXAMPLE 19 
A patient is diagnosed as suffering from a spinal trauma. The patient may 
then be administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that after 
the treatment the patient would be significantly protected from or would 
recover from ischemic injury resulting from the spinal trauma. 
EXAMPLE 20 
A patient is going to undergo surgery. The patient may then be administered 
an effective amount of the compounds of the present invention, such as set 
forth in example 6. It would be expected that after the treatment the 
patient would not develop an ischemic brain, spinal, or peripheral injury 
resulting from or associated with the surgery. 
EXAMPLE 21 
A patient is diagnosed as suffering from focal ischemia, such as that 
associated with thromboembolytic occlusion of a cerebral vessel, traumatic 
head injury, edema or brain tumors. The patient may then be administered 
an effective amount of the compounds of the present invention, such as set 
forth in example 6. It would be expected that after the treatment the 
patient would be significantly protected from or would recover from brain, 
spinal, or peripheral injury resulting from focal ischemia. 
EXAMPLE 22 
A patient is diagnosed as suffering from global ischemia. The patient may 
then be administered an effective amount of the compounds of the present 
invention, such as set forth in example 6. It would be expected that after 
the treatment the patient would be significantly protected from or would 
recover from a brain, spinal, or peripheral injury resulting from global 
ischemia. 
EXAMPLE 23 
A patient is diagnosed as suffering from a cardiac arrest. The patient may 
then be administered the compounds of the present invention, such as set 
forth in example 6. It would be expected that after the treatment the 
patient would be significantly protected from or would recover from an 
ischemic brain, spinal, or peripheral injury associated with cardiac 
arrest. 
EXAMPLE 24 
A patient is diagnosed as suffering from hypoxia, asphyxia or perinatal 
asphyxia. The patient may then be administered the compounds of the 
present invention, such as set forth in example 6. It would be expected 
that after the treatment the patient would be significantly protected from 
or would recover from an ischemic brain, spinal, or peripheral injury 
associated with the hypoxia, asphyxia or perinatal asphyxia. 
EXAMPLE 25 
A patient is diagnosed as suffering from a cerebro-cortical injury. The 
patient may then be administered the compounds of the present invention, 
such as set forth in example 6. It would be expected that after the 
treatment the patient would be significantly protected from or would 
recover from an ischemic brain injury resulting from the cerebro-cortical 
injury. 
EXAMPLE 26 
The patient is diagnosed as suffering from an injury to the caudate 
nucleus. The patient may then be administered the compounds of the present 
invention, such as set forth in example 6. It would be expected that after 
the treatment the patient would be significantly protected from or would 
recover from an ischemic brain injury resulting from the injury to the 
caudate nucleus. 
EXAMPLE 27 
A patient is diagnosed with a condition as shown in these examples. The 
compounds of the present invention may then be administered to the patient 
intravenously, intramuscularly, intraventricularly to the brain, rectally, 
subcutaneously, intranasally, through a catheter with or without a pump, 
placed adjacent or near tissue damaged by an ischemic event, orally, 
through a transdermal patch and/or topically, or through a polymer implant 
located adjacent to or near tissue damaged by an ischemic event. The 
patient's condition would be expected to improve. 
EXAMPLE 28 
A patient is diagnosed with a condition as shown in these examples. The 
compounds of the present invention may then be administered to the patient 
through a 100 mg/kg bolus. This may be followed by a 20 mg/kg intravenous 
infusion per hour over a two-hour period. The patient's condition would be 
expected to improve. 
EXAMPLE 29 
A patient is diagnosed with an cortical injury due to a condition such as 
set forth in these examples. The patient may then be administered the 
compounds of the present invention, such as set forth in example 6. It 
would be expected that the patient would be significantly protected from 
further injury, or would exhibit at least 65% to at least 80% recovery 
from the injury after treatment. 
EXAMPLE 30 
A patient is diagnosed with adenocarcinoma of the prostate. The patient may 
then be administered a NAALADase inhibitor, such as set forth in example 
6, by direct injection into the tumor. After this initial treatment, the 
patient may be optionally administered the same or different NAALADase 
inhibitor by intermittent or continuous administration by subdural pump. 
It would be expected that no further occurrences of the adenocarcinoma 
would develop. 
EXAMPLE 31 
A patient is diagnosed with adenocarcinoma of the prostate. The patient may 
then be administered a NAALADase inhibitor, such as set forth in example 
6, by direct injection into the tumor. After this initial treatment, the 
patient may be optionally administered the same or different NAALADase 
inhibitor by intermittent or continuous administration by implantation of 
a biocompatible, polymeric matrix delivery system. It would be expected 
that no further occurrences of the adenocarcinoma would develop. 
EXAMPLE 32 
A patient is diagnosed with benign prostatic hyperplasia. The patient may 
then be administered a NAALADase inhibitor, such as set forth in example 
6, by direct injection into the tumor. After this initial treatment, the 
patient may be optionally administered the same or different NAALADase 
inhibitor by intermittent or continuous administration by injection, 
subdural pump, or polymeric matrix implant. It would be expected that the 
benign prostatic hyperplastic cells do not develop into carcinoma. 
EXAMPLE 33 
A patient is diagnosed with adenocarcinoma of the prostate. The 
adenocarcinoma appears not to have metastasized. The adenocarcinoma would 
be removed by surgery. After post-operative recovery, the patient would be 
locally administered NAALADase inhibitor by intermittent or continuous 
administration by injection, subdural pump or by polymeric matrix implant. 
It would expected that no further occurrences of the carcinoma would 
develop. 
EXAMPLE 34 
A patient is diagnosed with metastatic adenocarcinoma of the prostate. The 
adenocarcinoma appears to have metastasized, but surgery still is 
indicated as an effective treatment modality. Tumor tissue would be 
removed by surgery. The patient would be locally administered a NAALADase 
inhibitor such as described herein from the time, approximately, of the 
initial diagnosis and would continue after surgery. After post-operative 
recovery, the patient would be maintained at this level of NAALADase 
inhibitor by a regimen of periodic local administration. The patient would 
be monitored carefully for intolerable adverse side-effects of NAALADase 
inhibitor administration. It would be expected that no further tumors 
develop. If some of the original, small tumorous masses are detected after 
surgery, they would be expected to not grow in size. 
EXAMPLE 35 
A patient is diagnosed with cancer as defined herein. The patient may then 
be administered a NAALADase inhibitor, such as set forth in example 6, by 
direct administration to the cancer cells. After this initial treatment, 
the patient may be optionally administered the same or different NAALADase 
inhibitor by direct injection, subdural pump, or implantation of a 
biocompatible, polymeric matrix delivery system. It would be expected that 
tumor growth or tumor cell growth would be prevented or inhibited and that 
no further occurrences of the cancer/tumor would develop. 
The invention being thus described, it will be obvious that the same may be 
varied in many ways. Such variations are not to be regarded as a departure 
from the spirit and scope of the invention and all such modification are 
intended to be included within the scope of the following claims.