U.S. patent application number 09/927207 was filed with the patent office on 2002-08-22 for selective inhibition of glutaminase by bis-thiadiazoles.
Invention is credited to Newcomb, Robert W., Newocmb, Marcell.
Application Number | 20020115698 09/927207 |
Document ID | / |
Family ID | 26918681 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115698 |
Kind Code |
A1 |
Newcomb, Robert W. ; et
al. |
August 22, 2002 |
SELECTIVE INHIBITION OF GLUTAMINASE BY BIS-THIADIAZOLES
Abstract
Compounds are disclosed which efficiently inhibit glutaminase
but which have no effect, at higher levels, on various
mechanistically and functionally related enzymes. The compounds,
which are useful for neuroprotection and in treatment of hepatic
encephalopathy, have the general formula I: 1 as defined further
herein.
Inventors: |
Newcomb, Robert W.; (Palo
Alto, CA) ; Newocmb, Marcell; (Whittier, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
26918681 |
Appl. No.: |
09/927207 |
Filed: |
August 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60224395 |
Aug 10, 2000 |
|
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Current U.S.
Class: |
514/363 |
Current CPC
Class: |
A61K 31/433
20130101 |
Class at
Publication: |
514/363 |
International
Class: |
A61K 031/433 |
Claims
It is claimed:
1. A method of selectively inhibiting glutaminase in a cell or
tissue, comprising administering to said cell or tissue an
effective amount of a compound of formula I: 4where X is sulfur or
oxygen, and R.sup.1 and R.sup.2 are independently selected from the
group consisting of lower alkyl, lower alkoxy, aryl, and
--(CH.sub.2 ).sub.n-aryl, where n is 0 or 1, and aryl is a
monocyclic aromatic or heteroaromatic group, having ring atoms
selected from the group consisting of carbon, nitrogen, oxygen, and
sulfur, and having at most three non-carbon ring atoms, which group
may be unsubstituted or substituted with one or more substituents
selected from halogen, lower alkyl, lower alkoxy, amino, lower
alkyl amino, amino(lower alkyl), or halo(lower alkyl).
2. The method of claim 1, wherein X is sulfur.
3. The method of claim 2, wherein each of R.sup.1 and R.sup.2 is
--(CH.sub.2).sub.n-aryl.
4. The method of claim 3, wherein each of R.sup.1 and R.sup.2 is
phenyl or benzyl, unsubstituted or substituted with lower alkyl or
lower alkoxy.
5. The method of claim 4, wherein each of R.sup.1 and R.sup.2 is
benzyl.
6. The method of claim 4, wherein each of R.sup.1 and R.sup.2 is
p-methoxy phenyl.
7. The method of claim 4, wherein each of R.sup.1 and R.sup.2 is
m-tolyl.
8. The method of claim 3, wherein each of R.sup.1 and R.sup.2 is
2-thiophenyl.
9. The method of claim 3, wherein each of R.sup.1 and R.sup.2 is
2-furanyl.
10. The method of claim 1, wherein each of R.sup.1 and R.sup.2 is
lower alkoxy.
11. The method of claim 10, wherein each of R.sup.1 and R.sup.2 is
ethoxy.
12. The method of claim 1, wherein each of R.sup.1 and R.sup.2 is
lower alkyl.
13. The method of claim 12, wherein each of R.sup.1 and R.sup.2 is
t-butyl.
14. The method of claim 1, wherein said tissue is neuronal tissue
having been subjected to ischemia, physical trauma, or a
neurodegenerative disorder.
15. The method of claim 1, wherein the tissue is intestinal tissue.
Description
[0001] This application claims the benefit of U.S. patent
application Ser. No. 60/224,395 filed Aug. 10, 2000, which is
incorporated in its entirety herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to selective and potent
inhibition of the enzyme glutaminase. Compounds are disclosed which
efficiently inhibit glutaminase but which have no effect, at higher
levels, on various mechanistically and functionally related
enzymes. The compounds are useful for neuroprotection and in
treatment of hepatic encephalopathy.
[0003] References
[0004] Ahluwalia, G. S. et al., Metabolism and action of amino acid
analog anti-cancer agents. Pharmac. Ther. 46:243-271 (1990).
[0005] Almeida, A. F. et al., Maximal activities of key enzymes of
glutaminolysis, glycolysis, krebs cycle and pentose-phosphate
pathway of several tissues in mature and aged rats. Int. J.
Biochem. 21:937-940 (1989).
[0006] Conjeevaram, H. S. et al., Reversal of behavioral changes in
rats subjected to portocaval shunt with oral neomycin therapy.
Hepatology 19:1245-1250 (1993).
[0007] Cooper, A. J. L., Ammonia metabolism in mammals: Interorgan
relationships, in Cirrhosis, Hyperammonemia, and Hepatic
Encephalopathy, Eds. Grisola, S., and Felipo, V., Plenum, N.Y.
(1994).
[0008] Di Pierro, D. et al., Analytical Biochemistry 231:407-412
(1995).
[0009] Dugan, L. L. et al., Glia modulate the response of murine
cortical neurons to excitotoxicity: Glia exacerbate AMPA
neurotoxicity. J. Neurosci. 15:4545-4555 (1995).
[0010] Hawkins, R. A. and Mans, A. M, Brain metabolism in hepatic
encephalopathy and hyperammonemia, in Cirrhosis, Hyperammonemia,
and Hepatic Encephalopathy, Grisola, S., and Felipo, V., Eds.,
Plenum, N.Y., pp. 13-19 (1994).
[0011] Kvamme, E. et al., Glutaminase from mammalian tissue. Meth.
Enzymol. 113:241-256 (1985).
[0012] Kerouel, R. and Aminot, A., Marine Chemistry 57:265-275
(1997).
[0013] Lo, E. H., Pierce, A. R., Matsumoto, K., Kano, T., Evans,
C., Newcomb, R., Alterations in K+ evoked profiles of
neurotransmitter and neuromodulator amino acids after focal
ischemia-reperfusion. Neuroscience 83:449-458 (1998).
[0014] Mousseau, D. D. and Butterworth, R. F., Current theories on
the pathogenesis of hepatic encephalopathy. Proc. Soc. Exp. Biol.
206(4):329-344 (1994).
[0015] Oppong, K. N. W. et al., Oral glutamine challenge in
cirrhotics pre- and post-liver transplantation: A psychometric and
analyzed EEG study. Hepatology 26:870-876 (1997).
[0016] Seiler, N. et al., Enhanced endogenous ornithine
concentrations protect against tonic seizures and coma in acute
ammonia intoxication. J. Pharmacol. Toxicol. 72:116-123 (1993).
[0017] Schousboe, A. et al., Preparation of primary cultures of
mouse (rat) cerebellar granule cells, in A Dissection and Tissue
Culture Manual of the Nervous System, Alan R. Liss, Inc., New York
(1989).
[0018] Shapiro, R. A. et al., Covalent interaction of
L-2-amino-4-oxo-5-chloropentanoic acid with rat renal
phosphate-dependent glutaminase. J. Biol. Chem. 253:7086-7090
(1978).
[0019] Shapiro, R. A. et al., Inactivation of rat renal
phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine.
Evidence for interaction at the glutamine binding site. J. Biol.
Chem. 254(8):2835-8 (1979).
[0020] Stauch, S. et al., Oral L-ornithine-L-aspartate therapy of
chronic hepatic encephalopathy: Results of a placebo-controlled
double-blind study. J. Hepatol. 28:856-864 (1998).
[0021] Wood, P., Roles of CNS macrophages in neurodegeneration, in
Neuroinflammation Mechanisms and Management, Wood, P. L., Ed.,
Humana, Totowa, N.J., pp 1-59 (1997).
[0022] Zea Longa, E. et al., Reversible middle artery occlusion
without craniectomy. Stroke 20: 84-91 (1989).
BACKGROUND OF THE INVENTION
[0023] Glutaminase is currently recognized as the most significant
glutamine utilizing enzyme present in mammalian central nervous
tissue (e.g., the brain and spinal cord). The enzyme catalyzes the
conversion of glutamine and water to glutamate, with the production
of ammonia.
[0024] Following ischemic insult or other traumatic injury to
neuronal cells, a number of biochemical changes occur in neuronal
tissue surrounding the injured region, including a rise in the
extracellular concentration of the excitatory neurotransmitter
glutamate. This high concentration of glutamate is believed to be
an important factor in delayed neuronal death, in which the
ischemic lesion increases approximately 2-fold over a period of
time 2-72 hours following the initial ischemic insult. It is
believed that this elevated glutamate level exacerbates the primary
insult, possibly by acting at excitatory glutamate receptors, and
that at least some of the excess glutamate results from enzymatic
conversion of glutamine to glutamate by glutaminase.
[0025] The maximal activity of glutaminase in the brain is 5-10
.mu.mol/min/g (Alameida et al., 1989). This activity corresponds to
a capacity for generating glutamate at a concentration of 5-10 mM
each minute, a rate far in excess of the 5-10 .mu.M IC.sub.50 for
toxicity of glutamate on isolated neurons (Dugan et al., 1995).
Therefore, it is apparent that only a very minor fraction of the
glutaminase present in the brain needs to be active in a
pathological circumstance in order to cause damage. Accordingly,
inhibition of glutaminase has been reported as a neuroprotective
treatment following ischemic injury (see e.g. Newcomb, PCT Pubn.
No. WO 99/09825).
[0026] Inhibition of glutaminase may also be used for treatment of
hepatic encephalopathy. This condition can arise from chronic liver
damage, such as chronic forms of viral hepatitis. Portal blood from
the intestine is shunted around the damaged liver, entering the
circulation directly. The resulting exposure of the brain to
elevated concentrations of blood ammonia produces neurologic
symptoms ranging from intellectual impairment and psychiatric
symptoms to coma. Several million people are affected to some
degree (Hawkins et al.). Current treatments are based on lowering
blood ammonia, either by decreasing ammonia generation in the gut,
e.g. by treatment with the antibiotic neomycin (Conjeevaram et
al.), or by biochemical manipulation of ammonia excretion. For
example, the efficacy of ammonia fixation by the urea cycle can be
increased by increasing the concentration of ornithine, which is
accomplished by inhibiting ornithine amino transferase with a
compound such as 5-fluoromethylornithine (Seiler et al.). The use
of L-ornithine-L-aspartate has also been reported (Stauch et al.).
These treatments, while useful, suffer from toxicity (neomycin) or
only partial effectiveness (ornithine compounds).
[0027] The role of glutaminase in regulation of blood ammonia is
described in Cooper et al. and Oppong et al., and includes the
hydrolysis of glutamine to glutamate and ammonia by glutaminase in
the intestine. Thus, hepatic encephalopathy could be treated by
selective inhibition of glutaminase in intestinal tissue.
[0028] To date, no glutaminase inhibitors have been reported that
are both potent and specific for glutaminase. Known inhibitors,
such as 6-diazo-5-oxo-L-norleucine ("DON"; Shapiro et al. 1979) and
L-2-amino-4-oxo-5-chloropentanoic acid ("chloroketone"; Shapiro et
al. 1978; see also Rosenberg, U.S. Pat. No. 5,156,976), also
inhibit a variety of glutamine utilizing enzymes, such as
amidotransferases (Ahluwahia et al., 1990).
SUMMARY OF THE INVENTION
[0029] The present invention includes, in one aspect, a method of
selectively inhibiting glutaminase in a cell or tissue, comprising
administering to the cell or tissue an effective amount of a
compound of formula I: 2
[0030] where
[0031] X is sulfur or oxygen, and R.sup.1 and R.sup.2 are
independently selected from the group consisting of lower alkyl,
lower alkoxy, aryl, and --(CH.sub.2 ).sub.n-aryl, where n is 0 or
1, and "aryl" is a monocyclic aromatic or heteroaromatic group,
having ring atoms selected from the group consisting of carbon,
nitrogen, oxygen, and sulfur, and having at most three non-carbon
ring atoms, which group may be unsubstituted or substituted with
one or more substituents selected from halogen, lower alkyl, lower
alkoxy, amino, lower alkyl amino, amino(lower alkyl), or halo(lower
alkyl). R.sup.1 and R.sup.2 may be the same or different; for ease
of preparation, R.sup.1 and R.sup.2 are the same.
[0032] Preferably, X is sulfur. In one embodiment, each of R.sup.1
and R.sup.2 is --(CH.sub.2 ).sub.n-aryl as defined above. The aryl
group may be carbocyclic, e.g. where each of R.sup.1 and R.sup.2 is
phenyl or benzyl, where the ring is unsubstituted or substituted as
recited above. Preferred compounds include those in which the ring
is unsubstituted or substituted with lower alkyl or lower alkoxy.
This group includes exemplary compounds in which each of R.sup.1
and R.sup.2 is benzyl, each of R.sup.1 and R.sup.2 is p-methoxy
phenyl, or each of R.sup.1 and R.sup.2 is m-tolyl. Compounds with
heterocyclic aryl groups include those in which each of R.sup.1 and
R.sup.2 is 2-thiophenyl or each of R.sup.1 and R.sup.2 is
p-furanyl. Compounds in which R.sup.1 and R.sup.2 are non-aryl
include those in which each of R.sup.1 and R.sup.2 is lower alkoxy,
such as ethoxy, or where each of R.sup.1 and R.sup.2 is lower
alkyl, e.g. t-butyl.
[0033] In one embodiment, the tissue is neuronal tissue which has
been subjected to ischemia, physical trauma, or a neurodegenerative
or neuropsychiatric disorder. Alternatively, the tissue is
intestinal tissue, particularly in a subject suffering from hepatic
encephalopathy.
[0034] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows a representative synthetic scheme for preparing
the compounds of the invention;
[0036] FIG. 2 shows inhibition of glutaminase activity in rat brain
membranes (PO.sub.4.sup.-3 conc.=8 mM) by invention compound
SNX-1770;
[0037] FIG. 3 shows the effect of glutamine on glutamate secretion
into medium by microglial (BV2) cells;
[0038] FIG. 4 shows inhibition of microglial glutamate secretion by
SNX-1770;
[0039] FIG. 5a shows the effect of SNX-1770 on intracellullar amino
acids in intestinal epithelial cells (CACO2);
[0040] FIG. 5b shows the effect of SNX-1770 on nucleotides in
intestinal epithelial cells (CACO2);
[0041] FIGS. 6a-c show dose response curves for lowering of
intracellular glutamate, aspartate, and .gamma.-amino butyric acid,
respectively, in cerebellar granule cells (day 4 cultures) and
astrocytes (day 12 cultures).
DETAILED DESCRIPTION OF THE INVENTION
[0042] I. Definitions
[0043] The terms below have the following meanings unless indicated
otherwise. "Alkyl" refers to a fully saturated acyclic monovalent
radical containing carbon and hydrogen, which may be branched or a
straight chain. Examples of alkyl groups are methyl, ethyl,
n-butyl, n-heptyl, and isopropyl. "Lower alkyl", a subset of this
class, refers to alkyl having one to six carbon atoms, and more
preferably one to four carbon atoms.
[0044] "Aralkyl" refers to a monovalent alkyl radical substituted
with an aryl group, as defined herein, e.g. a benzyl group
(--CH.sub.2C.sub.6H.sub.5).
[0045] A "pharmaceutically acceptable salt" of a compound described
herein refers to the compound in protonated form with one or more
anionic counterions, such as chloride, sulfate, phosphate, acetate,
succinate, citrate, lactate, maleate, fumarate, palmitate, cholate,
glutamate, glutarate, tartrate, stearate, salicylate,
methanesulfonate, benzenesulfonate, sorbate, picrate, benzoate,
cinnamate, and the like. Hydrochloride salts are a preferred group.
The term also encompasses carboxylate salts having organic and
inorganic cations, such as alkali and alkaline earth metal cations
(for example, lithium, sodium, potassium, magnesium, barium and
calcium); ammonium; or organic cations, for example,
dibenzylammonium, benzylammonium, 2-hydroxyethylammonium,
bis(2-hydroxyethyl) ammonium, phenylethylbenzylammonium,
dibenzylethylenediammonium, and the like. Such salts may be formed
by substitution of ionizable groups onto, for example, phenyl rings
in group R.sup.1 or R.sup.2, which can be useful for increasing
solubility or for reducing membrane permeability, if desired.
[0046] The terms "neuronal cell damage", "damage to neuronal
cells", and "cell injury" refer to conditions in which the
integrity of a neuronal cell has been compromised. This condition
may be a result of an ischemic event, a concussive traumatic event,
a degenerative event, or the like.
[0047] The terms "hypoxic", "hypoxia", "ischemic" and "ischemia",
as used herein, refer to conditions in which eukaryotic cells,
particularly neuronal cells, are exposed to oxygen concentrations
that are at least 50% less than a normal range of oxygen tension
required for normal growth and maintenance of such cells in culture
or in vivo.
[0048] II. Glutaminase Inhibiting Compounds
[0049] The selective glutaminase inhibiting compounds of the
invention have the general formula I: 3
[0050] where X is sulfur or oxygen, and R.sup.1 and R.sup.2 are
independently selected from the group consisting of lower alkyl,
lower alkoxy, aryl, and --(CH.sub.2).sub.n-aryl, where n is 0 or 1.
As used herein, "aryl" refers to a monocyclic aromatic or
heteroaromatic group, having ring atoms selected from the group
consisting of carbon, nitrogen, oxygen, and sulfur, and having at
most three non-carbon ring atoms. The aryl group may be
unsubstituted, or it may be substituted with one or more
substituents selected from halogen, lower alkyl, lower alkoxy,
amino, lower alkyl amino, amino(lower alkyl), and halo(lower
alkyl). Preferably, each ring has at most three substituents, more
preferably at most two, and most preferably one or no substituents.
While R.sup.1 and R.sup.2 are typically identical for ease of
synthesis, R.sup.1 and R.sup.2 may be different.
[0051] In preferred embodiments, X is sulfur, and each of R.sup.1
and R.sup.2 is --(CH.sub.2).sub.n-aryl, as defined above. In one
embodiment, each of R.sup.1 and R.sup.2 is phenyl or benzyl, where
the ring is unsubstituted or substituted with lower alkyl or lower
alkoxy. Preferably, the substituent is a single meta orpara
substituent. This group includes compounds designated herein as
SNX-1770 (unsubstituted benzyl), 1853 (unsubstituted phenyl) and
1832 (p-methoxy phenyl). Other preferred embodiments include
compounds in which X is sulfur and the aryl group is furanyl (e.g.
compound 1836) or thiophenyl (e.g. compound 1837).
[0052] Compounds of the invention may exist in other forms
depending on solvent, pH, temperature, and other variables known to
practitioners skilled in the art. For example, equilibrium forms
may include tautomeric forms. The compounds may be chemically
modified to enhance specific biological properties, such as
biological penetration, solubility, oral availability, stability,
metabolism, or excretion. The compounds may also be modified to
prodrug forms, such that the active moiety results from the action
of metabolic or biochemical processes on the prodrug.
[0053] The compounds can be prepared by reaction of a diacid, such
as shown in FIG. 1, with two moles of thiosemicarbazide to give a
bis(aminothiadiazole), followed by condensation with the
appropriate acid chloride(s) to give the desired R.sup.1 and
R.sup.2 substituents. Any reactive substituents that may be present
on the R.sup.1 or R.sup.2 groups may be protected, if necessary,
and then deprotected according to methods well known in organic
synthesis. Mixtures that may be produced when R.sup.1 and R.sup.2
are different are separated by known preparative methods, typically
by chromatography. Preparation of the representative compound
SNX-1770 (bis-2'-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethy- l
sulfide) is illustrated in FIG. 1 and described in Example 1.
[0054] III. Glutaminase Inhibiting Properties
[0055] A. Screening Methods
[0056] The compounds of the invention were evaluated based on their
ability to inhibit glutaminase in a cell-free assay (Example 2) and
in rat brain membranes (Example 3), and to inhibit glutamate
secretion by microglia (Examples 4-5). Effects of the compounds on
glutaminase were confirmed by assays of different cell types,
including neuronal cells and intestinal epithelial cells (Examples
6-7).
[0057] In general, glutaminase activity can be monitored by
detecting production of either of the products of the reaction,
i.e. glutamate or ammonia. Typically, glutamate production is
measured, since ammonia is a product of a number of other
biological reactions. Glutamate production can be measured by any
of a number of standard methods known in the art, including
chemical and chromatographic detection methods and coupled enzyme
assays that utilize NADH and glutamate dehydrogenase. Extracellular
glutamate concentrations can also be measured in vivo, using
microdialysis methods known in the art. One suitable method for
measuring glutamate is a microtiter-based two-step assay in which
glutamate formed in the initial step is quantitatively deaminated
by glutamate dehydrogenase to yield an equivalent amount of NADH
(Kvamme et al., 1985), which can then be detected
spectrophotometrically, as described in Example 2.
[0058] Specificity for glutaminase was evaluated at two levels.
Compounds were evaluated for specific interactions with several
thiol hydrolases (cathepsin B, caspase-9, calpain-1, and thrombin).
These are cysteine proteases which are believed to function by a
mechanism similar to that of glutaminase. Compounds were also
evaluated for effects on intracelluar amino acids and nucleotides
in CACO2 (intestinal epithelial) cells (Example 5), and for effects
amino acids in primary cultures of cerebellar neurons (granule
cells) and astrocytes (Examples 6-7). These measurements reflect
interactions with other glutamine utilizing enzymes involved in
amino acid and nucleic acid biosynthesis and homoeostasis. For
example, a decrease in cytidine triphosphate (CTP) and an increase
in uridine triphosphate (UTP) on treatment with DON
(6-diazo-5-oxo-L-norleuc- ine), a known nonselective antiglutamine
affinity label, is indicative of inhibition of the activity of CTP
synthetase, for which UTP is the substrate and CTP the product.
[0059] B. Cell Free Inhibition Assay
[0060] Compounds of formula I were tested for inhibition of human
kidney glutaminase as described in Example 2. Results are given in
Table 1 (IC.sub.50=concentration required for 50%
1 TABLE 1 Cmpd No. SNX- X.dbd. R.sup.1.dbd.R.sup.2 .dbd. IC.sub.50,
.mu.M 1770 S benzyl 0.19, 0.58 (two runs) 1830 S phenyl 1.06 1855 S
o-methoxy phenyl 4.90 1831 S m-tolyl 1.13 1832 S p-methoxy phenyl
0.46 1836 S 2-furanyl 1.12 1837 S 2-thiophenyl 0.39 1833 S methyl
4.42 1851 S t-butyl 1.65 1852 S ethoxy 1.78 1853 O phenyl 10.2
[0061] Compound 1770 gave similar results with enzyme expressed in
human kidney cells (IC.sub.50=0.18 .mu.M)and with native enzyme
(IC.sub.50=0.16 .mu.M).
[0062] C. Inhibition of Glutamate Secretion by Microglia
[0063] As described in Wood, glutamate efflux from microglia is
dependent on the presence of added external glutamine. Experiments
conducted in support of the present invention further showed that
microglia do not significantly take up or metabolize added
glutamate, and that glutamine dependent efflux is specific to
glutamate, linear with time, and inhibited by the antiglutamine
affinity label, 6-diazo-5-oxo-L-norleucine (DON). Therefore, the
concentration of glutamate in the culture medium can be used
directly as a measure of glutamate production by these cells.
[0064] In assaying the invention compounds, microglia (BV-2) cells
were cultured using standard methods, as described in Example 4. On
the day of the experiment, cells were incubated in glutamine free
medium for 1 to 1.5 hours, then glutamine and test compounds,
dissolved in glutamine free medium, were added. Aliquots were
removed at 2 h and 4 h for glutamate analysis. As shown in FIG. 4,
compound 1770 was able to inhibit glutamate efflux by microglia,
giving an IC.sub.50 value of 80-120 nM and ca. 85% inhibition at 10
.mu.M.
[0065] D. Effect on Levels of Intracellular Glutamate in Cells
[0066] The effect of compound 1770 on intracellular amino acids was
examined in CACO2 cells, granule cell neurons, and astrocytes, as
described in Examples 6 and 7, below. Effects on glutamate are
shown in FIGS. 5A and 6B. Compound 1770 caused a partial
(.about.30%) and specific lowering of cellular glutamate in all
cell types. IC.sub.50 was determined to be 100-300 nM in primary
cultures of astrocytes and 50-100 nM in primary cultures of
cerebellar granule cells.
[0067] E. Specificity: Effects on Other Intracellular Amino Acids
and Nucleotides
[0068] Compound 1770 had no effect at 10 .mu.M on a variety of
intracellular (non-glutamate) amino acids, including serine,
glycine, arginine, alanine, and taurine, in cultured cerebellar
neurons, astrocytes, and intestinal epithelial cells (Examples 6
and 7). This is illustrated in FIG. 5A for glycine and taurine. It
also had no effect on cellular GABA (gamma-amino butyric acid) in
granule cells at levels up to 10 .mu.M, as shown in FIG. 6C. (In
FIGS. 6A-C, data are expressed as the ratio of Glu, Asp, or GABA to
Ala (neurons and astrocytes) or Ser (astrocytes). Amounts of Ala
and Ser were shown not to change with SNX-1770.)
[0069] The remaining volume of the extracts was used for
chromatographic determination of nucleic acids, as described in Di
Pierro et al. This assay reflects the activity of a variety of
glutamine amidotransferases, including CTP synthetase, which are
mechanistically and functionally related to glutaminase.
[0070] CTP synthetase is a glutamine amidotransferase which
synthesizes CTP by adding the amide nitrogen of glutamine to UTP.
The enzyme is representative of a significant class of enzymes
which possess glutaminase activity, and which are thought to work
by a similar mechanism with the brain/kidney glutaminase, thus
providing a stringent test of specificity.
[0071] As shown in FIG. 5B, compound 1770 had no effect at 10 .mu.M
on intracellular nucleotides in intestinal epithelial cells (no
detectable difference between test culture and control). Nor did it
have any effect, at 30 .mu.M, on a variety of mechanistically
related enzymes, including thiol hydrolases, cathespin B, caspase
9, calpain 1, and thrombin (data not shown).
[0072] F. Effect on Ammonia Production by Intestinal Epithelial
Cells
[0073] As stated above, selected glutaminase inhibition in
intestinal tissue is expected to be beneficial in treating hepatic
encephalopathy, by lowering blood ammonia levels. To test the
effect of a compound on intestinal ammonia levels in vitro,
intestinal epithelial cells (e.g. CACO2) are incubated in a
balanced salt solution containing glucose and varying
concentrations of the compound, with or without glutamine (about 2
mM). Following incubation, aliquots of medium were removed, brought
to alkaline pH and placed in a closed vessel. The volatile ammonia
is trapped in a strong acid, typically in the form of drop hanging
at the top of the vessel, and, following neutralization, analyzed
fluorometrically with o-phthaldialdehyde and sulfite, by a
modification of the procedure of Kerouel and Aminot, 1997.
[0074] G. Cellular Permeability
[0075] Cellular permeability of SNX-1770 was demonstrated by its
ability to lower intracellular amounts of glutamate and the
biosynthetically related amino acids, at 30-100 nM in cultures of
cerebellar neurons and astrocytes, and at 10 .mu.M in cultures of
intestinal epithelial cells.
[0076] H. Low Toxicity
[0077] Compound 1770 showed no toxicity up to 100 .mu.M in cultured
retinal epithelial cells, as measured by an assay of mitochondrial
respiratory function. Administration of up to 60 mg/kg I.P. in rat
was well tolerated.
[0078] I. Noncompetitive Inhibition
[0079] Glutaminase inhibition by the compounds of the invention is
not competed by glutamine. The compounds bear no structural
similarity to either glutamine or glutamate. They are therefore
unlikely to interact with transporters, receptors or other enzymes
that recognize glutamine or glutamate, which is consistent with
their selectivity and low toxicity.
[0080] IV. Indications
[0081] A. Hepatic Encephalopathy
[0082] In accordance with the present invention, selective
inhibition of glutaminase in intestinal tissue can be used to treat
hepatic encephalopathy. As noted above, this is a condition in
which blood from the intestine is shunted around a damaged liver,
entering the circulation directly, and exposing the brain to
elevated concentrations of blood ammonia. It has been reported that
oral glutamine causes elevated blood ammonia in cirrhotics (Oppong
et al. 1998). Inhibition of intestinal glutaminase may thus be
directly therapeutic in hepatic encephalopathy.
[0083] Experiments with known inhibitors of glutamine-utilizing
enzymes in neurons and astrocytes were used, in support of the
invention, to establish that (1) glutaminase has a significant role
in reversing the net incorporation of ammonia into glutamine by
glutamine synthetase and that (2) steady state concentrations of
intracellular amino acids in neurons and astrocytes can be used as
a marker of nonselective inhibition of glutamine amidotransferase
activity. Similar procedures can be used to establish other cell
lines, such as intestinal epithelial cells, as model systems.
[0084] Cell based assays are used to measure the effect of
inhibitors on ammonia production by an intestinal cell line exposed
to glutamine, as described in Section IIIF above. Effects of
glutaminase inhibitors on intracellular amounts of a variety of
amino acids are also determined, as a measure of selectivity vs.
glutamine amidotransferases. Compounds with attractive profiles are
tested in one of the several animal models for hepatic
encephalopathy. The most common employs surgical shunting of
intestinal blood flow around the liver, or portacaval shunting
(Hawkins et al.; Conjeevaram et al.). Blood ammonia injections can
be given for comparative purposes. Compound effects are evaluated
biochemically, e.g. by measurement of blood ammonia and brain
glutamine, or behaviorally, e.g. by observing restoration of
locomotor activity (Hawkins et al.; Conjeevaram et al.).
[0085] Selective glutaminase inhibitors as described herein,
administered orally or intraperitoneally, are expected to
preferentially block ammonia generation by glutaminase in the
intestine. Inhibition of glutaminase in tissue such as kidney,
liver, and brain may provide additional lowering of blood levels of
ammonia. In addition, it is thought that alterations in amino acid
neurotransmission may underlie some of the neuropsychiatric
symptoms of hepatic encephalopathy. Glutaminase is also involved in
the biosynthesis of the brain neurotransmitter amino acids,
glutamate, aspartate, and .gamma.-amino butyrate. Accordingly,
intravenous administration may also be effective for treatment of
this disorder.
[0086] B. Neuroprotection
[0087] The compounds of the invention may be used to minimize
damage to neuronal tissue, such as may occur in the brain as a
result of cerebral ischemia, trauma, or degeneration. Ischemic
damage to the central nervous system (CNS) may result from either
global or focal ischemic conditions. 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. Ischemic diseases include cerebral
ischemia, such as results from stroke, myocardial infarction,
retinal ischemia, macular degeneration, and glaucoma.
[0088] Other sources of damage to central nervous tissue include
various neurodegenerative diseases such as Alzheimer's disease (Kim
et al., 1997), ALS and motor neuron degeneration (Greenlund et al.,
1995), Parkinson's disease (Ghosh et al., 1994), peripheral
neuropathies (Batistatou et al., 1993), Down's syndrome (Busciglio
et al., 1995), age related macular degeneration (ARMD) (Hinton et
al., 1998), Huntington's disease (Goldberg et al., 1996), spinal
muscular atrophy (Liston et al., 1996), and HIV encephalitis
(Lazdins et al., 1997).
[0089] Following ischemic insult or other traumatic injury to
neuronal cells, a number of biochemical changes occur in neuronal
tissue surrounding the injured region. Particularly relevant to the
present invention, there is a rise in the extracellular
concentration of the excitatory neurotransmitter glutamnate. It is
believed that this elevated glutamate level exacerbates the primary
insult, possibly by acting at excitatory glutamate receptors, and
that at least some of the excess glutamate results from enzymatic
conversion of glutamine to glutamate by glutaminase (Newcomb, WO
99/09825). This high concentration of glutamate is believed to be
an important factor in delayed neuronal death, in which the
ischemic lesion increases approximately 2-fold over a period of
time 2-72 hours following the initial ischemic insult. Newcomb also
disclosed that glutamine hydrolysis remains associated with cell
fragments after neuronal death, and showed that glutamate
generation by glutaminase in damaged neurons was sufficient to
account for glutamine toxicity in hypoxic neuronal/glial cells. As
shown herein, the invention compounds are able to block
pathological glutamate production in brain membranes and microglia,
the major sources of glutamate production in damaged brain
tissue.
[0090] A well-established in vivo model of cerebral ischemic damage
(stroke) is the rat middle cerebral artery occlusion (MCAO) model
in which a rat's middle cerebral artery is permanently occluded
(Zea Longa et al.). Such test paradigms are used to assess the
ability of test compounds to reduce neuronal damage in vivo. That
is, using the MCAO model as an example, animals subjected to MCAO
and fitted with a microdialysis probe in the affected brain region
are given a test glutaminase inhibiting compound through the probe.
Glutamate production is measured, as described above, and a
compound is considered to be potentially neuroprotective if it
attenuates the rise in glutamate in the ischemic penumbra
region.
[0091] Further tests are made giving the test compound
systemically, as this mode of administration is contemplated for
use in the treatment of mammalian subjects. That is, a compound is
given systemically, typically intravenously, to test animals who
have undergone neuronal insult, such as ischemic insult to the
central nervous system, as discussed above. The compound is
administered in a pharmaceutically acceptable vehicle, preferably
an aqueous vehicle, such as normal or buffered saline. For
compounds with low aqueous solubility, suspensions of very fine
particles have been employed. Selected brain regions are then
assessed for presence of absence of neuronal damage, usually by any
of a number of histological techniques known in the art. Direct
administration to the affected region, such as by
intracerebroventricular or intrathecal delivery routes or direct
shunt, are also possible in the context of the present
invention.
[0092] Dose-response studies, which are within the knowledge and
judgement of skilled practitioner, can be used to establish
adequate dosing ranges for experimental treatment paradigms, as
well as to extrapolate such doses to use in larger animals,
including humans.
[0093] C. Neuropsychiatric Disorders and other Disorders of
Neuronal Signaling
[0094] The compounds of the invention may also be used for
regulation of neurotransmission, i.e. in the treatment of
neuropsychiatric disease. Glutaminase participates, directly or
indirectly, in the biosynthesis of the major excitatory
neurotransmitters of brain, glutamate and aspartate, and the main
inhibitory neurotransmitter, .gamma.-amino butyrate. Partial
inhibition of glutaminase in intact nervous tissue may be used to
modulate the balance of excitatory and inhibitory
neurotransmission, and thus be of value in treating disorders of
nervous system function which involve aberrant neuronal signaling.
These disorders include pain, epilepsy, and neuropsychiatric
disorders such as depression, anxiety and schizophrenia.
EXAMPLES
[0095] The following examples illustrate but are not intended in
any way to limit the invention.
Example 1
[0096] Synthesis of SNX-1770
(Bis-2'-[5-(phenylacetamido)-1,3,4-thiadiazol- -2-yl]ethyl
sulfide)
[0097] Into a 1L pear-shaped flask were place 9.1 g
thiosemicarbazide (H.sub.2N(C.dbd.S)NHNH.sub.2), 8.9 g
3,3'-thiodipropionic acid (see FIG. 1), and 90 POCl.sub.3. The
resulting suspension was heated at 90.degree. C. for 3 h, then
cooled to room temperature and poured into 400 g of ice. The
resulting mixture was filtered and then brought to pH 14. The white
solid which formed was washed with 2.times.200 mL water and dried
in vacuo at 50.degree. C. to give 8 g
bis-2'-(5-amino-1,3,4-thiadiazol-2-yl) ethyl sulfide (see FIG. 1).
This product (4.32 g) was heated with 24 mL pyridine and 4.5 mL
phenyl acetyl chloride until the mixture was homogeneous. The
solution was cooled to room temperature and triturated with 50 mL
methanol and filtered to give a crude solid. This solid was
redissolved in 8 mL DMSO, 50 mL methanol was added, and the
solution was allowed to sit at room temperature as the product
crystallized. The product was collected and dried in vacuo at
50.degree. C. to give approx. 1.5 g of the product SNX-1770.
Example 2
[0098] Glutaminase Inhibition: Cell Free Assay
[0099] A. Enzyme Preparation
[0100] Glutaminase gene-transfected SF9 cells were collected in a
50-mL polypropylene conical tube by centrifugation at 500.times.g
for 10 min (Eppendorf Centrifuge 5810R, F34-6-38 rotor). The
supernatant was discarded, and the cell pellet was stored at
-80.degree. C. For preparation of the lysate, the cells were thawed
on ice and resuspended by pipetting in 0.8 mL cold lysis buffer
with 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a
serine protease inhibitor. The suspension was stored in ice for 20
min, then subjected to two freeze-thaw cycles (liquid nitrogen
followed by a room temperature water bath).
[0101] The volume of the cell homogenate was measured, and 1/3
volume of 1 M sucrose was added, to give a final concentration of
0.25 M sucrose. Aliquots 100 .mu.L in volume were placed in 1.5 mL
microtubes and stored at -80.degree. C.
[0102] Alternatively, HEK (human kidney) cells were used. The
medium was removed by aspiration, 5 mL PBS/1 mM EDTA was added, and
the culture was incubate for 3 min to allow cells to detach. The
cells were collected in a 50-mL conical tube using Eppendorf
Centrifuge 5810R, F34-6-38 rotor, and spun at 200.times.g for 5
min. The supernatant was discarded, and the cell pellet was
resuspended pipetting (approximately 0.12 g wet weight) in 0.3 mL
lysis buffer with 0.5 mM AEBSF. The tube was left in ice for 20
min. The suspension was stored in ice for 20 min, then subjected to
two freeze-thaw cycles as described above. Aliquots 100 .mu.L in
volume were placed in 1.5 mL microtubes and stored at -80.degree.
C.
[0103] The activity of the enzyme was determined colorimetrically.
One unit of enzyme activity is defined as the amount of enzyme
required to generate net OD 540 nm=0.6 in a total reaction volume
of 220 .mu.L in one hour at room temperature.
[0104] B. Assay Procedure
[0105] Assay plates were prepared containing 2 .mu.L test compound
in DMSO/well. The enzyme was diluted to 1 unit (liver) or 0.8 unit
(kidney)/100 .mu.L in glutaminase assay buffer, and 100 .mu.l
diluted enzyme was added to each well of the assay plate by
Multidrop (Labsystems). The contents were mixed by shaking at fall
speed for 1 min on TiterMix 100 (Brinkmann). The plates were
preincubated at room temperature (RT) for 20 min to allow binding
of test compounds to glutaminase, and 50 .mu.L glutamine solution
(7 mM in assay buffer) was added to each well by Multidrop. The
contents were shaken at full speed for 30 sec on TiterMix 100
(Brinkmann), and the plates were then incubated at RT for 60 min
(liver) or 90 min (kidney). To stop the reactions, 20 .mu.L HCl
(0.3 N) was added to each well by Multidrop and mixed immediately
by shaking for 30 sec on TiterMix 100.
[0106] For quantification, glutamate (formed by
glutaminase-catalyzed hydrolysis of glutamine) is oxidized to
2-oxoglutarate by a second enzyme, glutamate dehydrogenase (GDH),
with the concomitant production of the reduced form of nicotinamide
adenine dinucleotide (NADH). Reduction of nitro blue tetrazolium
(NBT) in the assay solution by NADH, catalyzed by phenazine
methosulphate (PMS), results in the formation of a blue-purple
formazan. The absorption of formazan at 540 nm is linearly
proportional to the concentration of glutamate up to 200 .mu.M.
[0107] NBT/GDH reagent (50 .mu.l) was added to each well by
Multidrop and mixed by shaking for 30 sec on TiterMix 100, and the
plates were incubated at RT for 20 min to allow color formation by
the GDH reaction. Glutamate concentration was determined from
formazan concentration as determined by reading OD540 nm on a
SpectraMax 340.
Example 3
[0108] Inhibition of Glutaminase Activity in Rat Brain
Membranes
[0109] Rat brain membranes in 0.4 .mu.L of 330 mM sucrose/20 mM
tris (pH 8.6) were added to 40 .mu.L of an aqueous solution
containing glutamine, sodium phosphate, pH 8.6, and 5 .mu.L of
SNX-1770 diluted from a 10 mM stock DMSO solution. The final assay
concentrations were 5.7 mM glutamine; 1, 8 and 160 mM phosphate;,
and 0.33 pM to 3.3 .mu.M SNX-1770. Aliquots of 10 .mu.L were
removed at 0, 85, and 195 min, added to 90 .mu.L 1:3 DMF/H.sub.2,
and analyzed for glutamate by HPLC following derivatization with
o-phthaldialdhyde and 2-mercaptoethanol (see Lo et al. 1998).
Results are shown in FIG. 2.
Example 4
[0110] Effect of Glutamine on Glutamate Secretion by Microglial
(BV2) Cells
[0111] BV-2 cells were cultured using Dulbeccos minimal essential
medium containing 10% heat inactivated fetal bovine serum, and
grown to 70-80% confluency before use. On the day of the
experiment, cells were placed in glutamine free medium and aliquots
removed for glutamate analysis at 0, 10 and 60 min. Glutamine was
then added, and further aliquots removed for glutamate analysis, as
above, at each of the next two hours. Results are shown in FIG.
3.
Example 5
[0112] Inhibition of Microglial Glutamate Secretion
[0113] BV-2 cells were cultured in 24 well plates using Dulbeccos
minimal essential medium containing 10% heat inactivated fetal
bovine serum, and grown to 70-80% confluency before use. On the day
of the experiment, cells were placed in glutamine free medium for 1
to 1.5 hours. Glutamine and SNX-1770 (diluted from a 10 mM stock
DMSO solution) were dissolved in glutamine free medium at two times
the test concentration (i.e., 2 mM for glutamine). This solution
was used to replace one half of the 0.5 mL culture medium volume at
the end of the glutamine free incubation. Aliquots of 10 .mu.L
medium were removed at 2 h and 4 h and placed in 96 well microtiter
plates with 90 .mu.L DMF/water (1:9) for glutamate analysis.
Results are shown in FIG. 4. Compound 1770 was able to inhibit
glutamate efflux by microglia, giving an IC.sub.50 value of 80-120
nM and ca.85% inhibition at 10 .mu.M.
Example 6
[0114] Effect of SNX-1770 on Intracellullar Amino Acids and
Nucleotides in Intestinal Epithelial Cells
[0115] Intestinal epithelial (CACO2) cells were grown in 12 well
plates with Eagles minimal essential medium containing 2 mM
glutamine, as recommended (American Tissue Culture Collection).
When the cells reached about 80% confluency, fresh glutamine was
added to 2 mM. SNX-1770 was diluted from a 100 .mu.M stock in
medium and added at concentrations varying from 10 nM to 10 .mu.M.
After 6.5 h of incubation, cultures were washed twice with
phosphate buffered saline and extracted with 100 .mu.L 1M
perchloric acid. Extracts were neutralized with 30 .mu.L of 5M
potassium carbonate, centrifuged, and analyzed by HPLC for
nucleotides, as described in Di Pierro et al. 1995, and for amino
acids, as described in Lo et al. 1998. Results are shown in FIG. 5A
(amino acids) and FIG. 5B (nucleotides).
[0116] FIG. 5A compares the effects of differing doses of SNX-1770
on glutamate, glycine and taurine in the extracts, showing a dose
dependent decrease in glutamate, with no effect on glycine or
taurine (data are n=3 analyses of 3 .mu.L aliquots from independent
extracts of independent culture wells, .+-.std. dev.). FIG. 5B
shows analysis for nucleotides (n=1 analysis of pooled extracts
from 3 culture wells exposed to control medium [top], or medium
containing 10 .mu.M SNX-1770).
Example 7
[0117] Effects of SNX-1770 on Intracellular Amino Acids in Primary
Cultures of Cerebellar Granule Cells
[0118] Primary cultures of cerebellar cortex were prepared in 6
well plates with 2.5 mL Dulbeccos minimal essential medium/horse
serum, as described in A. Schousboe et al., 1989. At day 3, when
cultures contained primarily the granule cell neurons, either 2.5
.mu.L of DMSO or 10 mM SNX-1770 in DMSO (final concentration, 10
.mu.M) was added to individual wells. After 5 h incubation, the
cultures were washed twice with phosphate buffered saline,
extracted with perchloric acid and analyzed for amino acids as
described for FIG. 5A.
[0119] To produce dose response curves for lowering of
intracellular Glu and Asp (FIG. 6A-B), the procedure was repeated
with 4 days (for cerebellar granule cells) and 12 days of
incubation (for astrocytes). A series of dilutions of SNX-1770 were
made in DMSO, and these were added to 4 or 7 culture wells (at days
4 and 12, respectively), such that wells contained 0.1% DMSO and/or
concentrations of SNX-1770 from 10 nM to 10 .mu.M. Results are
shown in FIGS. 6A-B. Data are expressed as the ratio of Glu, Asp,
or GABA to Ala (neurons and astrocytes) or Ser (astrocytes).
(Amounts of Ala and Ser do not change with SNX-1770.)
[0120] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications may be made without departing from the
invention.
* * * * *