U.S. patent application number 12/779566 was filed with the patent office on 2010-09-30 for neurotherapeutic composition and method therefor.
Invention is credited to Michael O. Chaney, Gary A. Koppel.
Application Number | 20100249090 12/779566 |
Document ID | / |
Family ID | 25128486 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100249090 |
Kind Code |
A1 |
Koppel; Gary A. ; et
al. |
September 30, 2010 |
NEUROTHERAPEUTIC COMPOSITION AND METHOD THEREFOR
Abstract
Neurotherapeutically effective pharmaceutical compositions are
described that include carboxypeptidase E inhibitors. One class of
carboxypeptidase E inhibitors found to exhibit significant
neurotropic activity are .beta.-lactam compounds, particularly
penam and cephem .beta.-lactam antibiotics and non-antibiotic
derivatives thereof.
Inventors: |
Koppel; Gary A.;
(Indianapolis, IN) ; Chaney; Michael O.; (Carmel,
IN) |
Correspondence
Address: |
BARNES & THORNBURG LLP
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Family ID: |
25128486 |
Appl. No.: |
12/779566 |
Filed: |
May 13, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11711971 |
Feb 28, 2007 |
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12779566 |
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10224124 |
Aug 20, 2002 |
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11711971 |
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09783201 |
Feb 14, 2001 |
6489319 |
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10224124 |
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Current U.S.
Class: |
514/210.08 ;
514/210.05; 514/210.06 |
Current CPC
Class: |
A61K 31/546 20130101;
A61K 31/5383 20130101; A61K 31/536 20130101; A61P 25/00 20180101;
A61K 31/197 20130101; A61K 31/4353 20130101; A61K 31/431 20130101;
A61K 31/545 20130101; A61K 31/5365 20130101; A61K 31/198 20130101;
A61K 31/43 20130101; A61K 31/424 20130101; A61P 25/22 20180101;
A61K 45/06 20130101; A61K 31/00 20130101; A61K 31/395 20130101;
A61K 31/197 20130101; A61K 2300/00 20130101; A61K 31/198 20130101;
A61K 2300/00 20130101; A61K 31/395 20130101; A61K 2300/00 20130101;
A61K 31/424 20130101; A61K 2300/00 20130101; A61K 31/43 20130101;
A61K 2300/00 20130101; A61K 31/431 20130101; A61K 2300/00 20130101;
A61K 31/4353 20130101; A61K 2300/00 20130101; A61K 31/5365
20130101; A61K 2300/00 20130101; A61K 31/5383 20130101; A61K
2300/00 20130101; A61K 31/545 20130101; A61K 2300/00 20130101; A61K
31/546 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/210.08 ;
514/210.05; 514/210.06 |
International
Class: |
A61K 31/5365 20060101
A61K031/5365; A61P 25/00 20060101 A61P025/00; A61P 25/22 20060101
A61P025/22; A61K 31/43 20060101 A61K031/43; A61K 31/424 20060101
A61K031/424 |
Claims
1. A neurotherapeutic pharmaceutical composition in unit dosage
form comprising a neurotherapeutically effective amount of a
carboxypeptidase E inhibitor and a pharmaceutically acceptable
carrier thereof; providing that when the carboxypeptidase E
inhibitor is a .beta.-lactam antibiotic, the neurotherapeutically
effective amount of the carboxypeptidase E inhibitor is less than
an antibiotically effective amount of the carboxypeptidase E
inhibitor if such .beta.-lactam antibiotic were to be administered
in a unit dosage form by the same route of administration.
2. The neurotherapeutic pharmaceutical composition of claim 1
wherein the neurotherapeutically effective amount is an amount
effective for treating behavioral disorders or enhancing cognitive
function in patients in need of such therapy.
3. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a compound comprising a
.beta.-lactam ring structure.
4. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is selected from the group
consisting of penams, cephems, 1-oxa-1-dethia cephems, clavams,
clavems, azetidinones, carbapenams, carbapenems and
carbacephems.
5. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a penam or cephem
compound.
6. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a cephem sulfoxide or
cephem sulfone compound.
7. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a cephem sulfoxide
compound.
8. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a cephem sulfone
compound.
9. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a penam sulfoxide or
penam sulfone compound.
10. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a penam sulfone
compound.
11. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a penam sulfoxide
compound.
12. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a penicillin or
cephalosporin.
13. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a sulfoxide or sulfone
derivative of a penicillin or of a cephalosporin.
14. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a 1-dethia-1-oxa-cephem
compound.
15. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is a compound of the
formula ##STR00005## wherein R is a salt forming group or an active
ester forming group; R.sub.1 is hydrogen or C.sub.1-C.sub.4 alkoxy;
X is S, SO, SO.sub.2, O, or CH.sub.2; and T is C.sub.1-C.sub.4
alkyl, halo, hydroxy, O(C.sub.1-C.sub.4) alkyl, or --CH.sub.2B
wherein B is the residue of a nucleophile B:H.
16. The neurotherapeutic pharmaceutical composition of claim 1
wherein the carboxypeptidase E inhibitor is moxalactam or a
pharmaceutically acceptable salt or ester thereof.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/783,201, filed Feb. 14, 2001, which is
expressly incorporated by reference herein.
FIELD OF INVENTION
[0002] This invention relates to a novel mechanism of
neuropsychiatric intervention. More particularly, this invention is
directed to pharmaceutical formulations and methods for treatment
of a variety of neurological disease states, including cognitive
and behavioral disorders.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] The pharmaceutical industry has directed extensive research
and development efforts toward discovery and commercialization of
drugs for treatment of neurological disorders. Such disorders
typically derive from chemical imbalances in the brain.
Overproduction or underproduction of pertinent neurochemical
species and/or receptor dysfunction has been identified with many
disease states recognized by neurologists, psychiatrists,
psychologists and other medical practitioners skilled in the
diagnosis and treatment of mental disease. Most of the discovery
effort for new neurologically active drugs has been based on the
study of agonist/antagonist drug interaction with one or more of
the numerous receptors in the brain and/or their respective
receptor ligands.
[0004] The present invention provides a novel approach to drug
intervention in the treatment of a wide variety of neurologic
disease states and other disease states or clinical conditions of
related etiology. It is based in part on the discovery that
.beta.-lactam containing compounds known for their activity as
inhibitors of bacterial peptidases or proteases, particularly
transpeptidases and/or carboxypeptidases, are also inhibitors of
certain mammalian neuro-peptidases, including
N-acetylated-.alpha.-linked acidic peptidases (NAALADases), several
of which have been identified/characterized in the literature
[Pangalos et al., J. Biol. Chem., 1999, 274 No. 13, 8470-8783]. The
present invention is also based in part on the discovery that
neurogenic carboxypeptidases can be targeted with carboxypeptidase
inhibitors to effect significant behavioral modification and
enhanced cognitive performance. Preliminary studies have confirmed
that one or more neurogenic proteases, now believed to be
NAALADases and related peptidases and transferases, capable of
recognizing and transforming certain neuropeptides (e.g.,
N-acetyl-L-aspartyl-L-glutamate) play a significant if not dominant
role at the neurochemical level of brain function and concomitantly
have a substantial impact on patient behavior and cognitive
performance. It has been previously reported that certain glutamate
analogs acting as NAALADase inhibitors can be used to treat
prostate disease and glutamate abnormalities associated with
certain nervous tissue insult. It has now been determined that
certain .beta.-lactam-containing bacterial peptidase inhibitors
capable of blood-brain barrier transport, can function in the brain
at very low concentrations as potent neuroactive drug substances to
reduce the symptoms of a wide variety of neurological disorders
characterized by behavioral aberration or sensory/cognitive
dysfunction. Significantly, such bacterial enzyme inhibitors are
believed to be effective inhibitors of neurogenic peptidases,
particularly carboxypeptidase E, at concentrations below those
concentrations known to be required for clinically effective
bacterial enzyme inhibition.
[0005] Accordingly, one embodiment of the present invention is
directed to a method for treatment of cognitive and behavioral
disorders in warm-blooded vertebrates by administering compounds
known for their activity as bacterial protease or peptidase
inhibitors, which compounds, when present at effective
concentrations in the brain, have now been determined to be capable
of inhibiting or otherwise modulating the activity of one or more
neurogenic enzymes.
[0006] In a related embodiment there is provided method for
treatment of cognitive and behavioral disorders in a patient in
need of such treatment. The method comprises the step of inhibiting
neurogenic peptidases, particularly, carboxypeptidase E and related
neurogenic enzymes. Such neuropeptidase inhibition is effected by
administering an effective amount of a .beta.-lactam compound
recognized for its capacity to bind to and inhibit a bacterial
enzyme, for example, a .beta.-lactamase or a bacterial protease
involved in bacterial cell wall synthesis. Such bacterial proteases
are known in the art as "penicillin binding proteins." Exemplary of
.beta.-lactam compounds for use in this invention are moxalactam,
its salts, esters and structurally related cephems and
1-oxa-1-dethia cephems. Effective inhibition of
neurogenic-carboxypeptidase E and related neuro-peptidase activity
in warm-blooded vertebrates in accordance with this invention has
been found to produce marked enhancement in cognitive performance
and behavioral management.
[0007] Exemplary of cognitive and behavioral disorders susceptible
to treatment in accordance with this invention include aggressive
disorder, obsessive compulsive disorder, anxiety, depression, ADHD,
and memory impairment. Animal data suggest that the method and
formulation of this invention have potential as an antiaggressive
agent to control impulsivity and violence in autism, Tourette's
syndrome, mental retardation, psychosis, mania, senile dementia and
individuals with personality disorders and history of inappropriate
aggression. Clinic applications extend to the treatment of children
with ADHD and conduct disorder, as an anxiolytic, and as a
cognition enhancer for the geriatric population to improve learning
and memory and to ameliorate disorientation.
[0008] In another embodiment of this invention there is provided a
method of treating a patient afflicted with a condition, or
disposed to development of a condition, characterized at least in
part by abnormal extracellular concentration of glutamate in the
brain or other nervous tissue. The method comprises the step of
administering to the patient in effective amounts of a compound
capable of inhibiting the activity of a penicillin-binding protein
of bacterial origin. The composition is administered in an amount
effective to prevent or alleviate the symptoms of such condition.
The method and formulation embodiments of the invention find use in
both human health and veterinary applications, e.g., in canine,
feline and equine species.
[0009] In one embodiment of the present invention a warm-blooded
vertebrate, most typically a human patient, affected by a
neurologic disease state characterized by cognitive or behavioral
abnormalities is treated with a 1-oxa-1-dethia cephalosporin, more
preferably a 7-methoxy-1-oxa-1-dethia cephalosporin, optionally as
an active ester derivative in an orally (including buccal or
sublingual administration) or a parenterally administered
formulation. In one embodiment, the peptidase inhibitor is
moxalactam,
[7-.beta.-[2-carboxy-2-(4-hydroxyphenyl)acetamido]-7.alpha.-methoxy-3-[[(-
1-methyl-1H-tetrazol-5-yl)thio]methyl]-1-oxa-1-dethia-3-cephem-4-carboxyli-
c acid], described and claimed with related compounds, including
their orally absorbed active ester derivatives, in U.S. Pat. No.
4,323,567, the specification of which is expressly incorporated
herein by reference. Moxalactam has been found to exhibit
significant dose responsive neuroactivity when administered
parenterally at least at about 50 .mu.g/kg of body weight.
[0010] In another embodiment of the present invention there is
provided a pharmaceutical formulation for treatment with consequent
reduction of symptoms of behavioral or cognitive disorders in
patients in need of such treatment. The formulation comprises a
compound characterized not only by its affinity to bacteria derived
penicillin-binding proteins, but as well, its affinity to
neurogenic carboxypeptidases, particularly carboxypeptidase E. In
that embodiment the level of activity exhibited by the
carboxypeptidase inhibitor in the present method is not only
dependent on its affinity to penicillin-binding proteins and to
carboxypeptidase, namely carboxypeptidase-E, it is also
particularly dependent on ability of the inhibitor compound to
cross the blood brain barrier to achieve levels in the brain
effective to modify patient behavior and/or cognitive performance.
While the formulations of this invention can be prepared
specifically for any art-recognized mode of administration capable
of achieving threshold minimum protease inhibiting concentrations
in the brain, they are typically formulated for parenteral or oral
administration, optionally in the form of prolonged release or
"drug depot" type formulations well known in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1-42 are graphic representations of data gathered in
the conduct of testing of moxalactam, other .beta.-lactam
antibiotics, clavulanic acid and other neuroactive compounds in
various animal models accepted in the art for detection of activity
against offensive aggression (FIGS. 1-4, 9-14, 24, 29, 31 and 32),
general motor activity, olfactory discrimination (FIG. 5), sexual
activity (FIG. 6), anxiolytic activity (FIGS. 7, 25, 26, 28, 37 and
40), and spatial memory (FIGS. 8 and 29-36). FIGS. 15 and 16 are
graphic illustrations of the effect of intracerebrally administered
peptidoglycan-precursor protein on offensive aggression and
olfactory discrimination in hamsters.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention and the various embodiments described
and claimed herein derive, in part, from the discoveries that
compounds capable of binding to and inhibiting enzyme activity of
penicillin-binding proteins of bacterial origin are also potent
inhibitors of N-acetylated-.alpha.-linked acid dipeptidase
(NAALADase) activity and other related enzymes in the brain,
including carboxypeptidase E, and that when administered to provide
effective threshold enzyme inhibitory concentrations of same in the
brain, such inhibitors exhibit clinically significant neuroactivity
evidenced in part by behavioral modification and enhanced cognition
and function.
[0013] In one embodiment the peptidase inhibitors effective for use
in accordance with the present invention are characterized by their
capacity to inhibit a bacterial protease exhibiting selective
proteolytic activity on a protein or peptide substrate comprising
acyl-D-alanyl-D-alanine. Alternatively stated, effective inhibitors
for use in treatment of behavioral and cognitive disorders in
accordance with one embodiment of this invention, can be
characterized by their selective affinity (by associative and/or
covalent binding) to penicillin-binding proteins; such compounds
include particularly .beta.-lactam antibiotics such as penicillins,
cephalosporins and analogues thereof, particularly
1-oxa-1-dithiacephems. Based on animal tests to date, such
bacterial protease inhibitors appear to function at
subclinical-antibiotic levels in the brain to inhibit
neuropeptidase activity which can function in neurochemical
mediation of behavior and cognitive performance. Effective
inhibition of neuropeptidase activity with concomitant mediation of
behavior and cognitive performance has also been effected by
administration of a .beta.-lactamase inhibitor, clavulanic acid, a
.beta.-lactam containing compound having no clinically significant
antibiotic activity. It is surmised that inhibition of
neuropeptidase activity allows modulation of the concentration
and/or function of one or more neurotransmitters or neuromodulators
with concomitant improvement in neurological function evidenced by
enhancement of cognitive performance and attenuation of aberrant
behavioral phenotypes.
[0014] In one example of this invention, moxalactam given i.p. at
50 micrograms/kg inhibits aggression in hamsters, enhances spatial
learning in rats, and acts as an anxiolytic in rats. Clavulanic
acid has shown anxiolytic activity when administered i.p. at less
than 1 microgram/kg; it also exhibits potent neuroprotectant
activity, but animal testing data available for clavulanic acid to
date do not evidence the potent antiaggression and cognition
enhancement activity exhibited by moxalactam (although clavulanic
acid did exhibit a modest level of such activities). Nor does
moxalactam appear to exhibit the neuroprotectant activity seen for
clavulanic acid. The unique neurological activity profiles of
clavulanic acid and moxalactam provides strong evidence that the
compounds are each interacting with unique sets of neurogenic
targets.
[0015] Historically, those knowledgeable in the field of beta
lactam antibiotics understand that the mode of action as
antibacterial agents is by inhibiting cell wall synthesis by acting
as a substrate for penicillin-binding protein (PBP); the term PBP
has been extended to include binding to all beta lactams including
cephalosporins. More recently, investigators have been able to
clone and sequence these PBP's as well as crystallize the enzymes
and determine active site motifs (see P. Palomeque et al., J.
Biochem., 279, 223-230, 1991). Based on this data, the four
putative binding sites for PBP have been designated sites I, II,
III and IV. The sites, sequence location and amino acid (AA)
sequence are as follows:
TABLE-US-00001 Site I: 35 AA's downstream from N-terminus: STTK
(SEQ ID NO: 1) Site II: 57 AA's downstream from STTK (SEQ ID NO: 1)
motif: SGC, SGN, or SAN Site III: 111 AA's downstream from SGC
motif: KTG Site IV: 41 AA's downstream from SGC motif: ENKD (SEQ ID
NO: 2)
[0016] Pursuant to identifying an enzyme system in the brain that
moxalactam would inhibit in a similar manner to PBP, it was
discovered that a glutamyl carboxypeptidase enzyme known as
N-acetyl-.alpha.-linked acidic dipeptidase (NAALADase) (See M. N.
Pangalos et al., J. Bio. Chem., 264, 8470-8483, 1999) has an almost
perfect overlap of the putative active sites of PBP. This enzyme
system is responsible for regulating the glutamatergic
neurotransmission pathways, the effects of which would be expressed
in such behavioral outcomes as aggression, memory/cognition, and
anxiety. As a result of the almost perfect overlap of the putative
active sites of PBP and the conserved sequences in human and rat
NAALADase, it was initially believed that moxalactam and other
.beta.-lactam compounds mediate behavioral effects by inhibiting
NAALADase at low concentrations. This was based on the following
overlap sequence similarity between PBP and NAALADase I, one of
several known NAALADase species, as shown below:
TABLE-US-00002 Site I: PBP: 35 AA's downstream from N-terminus:
STTK (SEQ ID NO:1) NAALADase: 38 AA's downstream from N-terminus:
STQK (SEQ ID NO: 3) Site II: PBP: 57 AA's downstream from STTK (SEQ
ID NO: 1) motif: SGC, SGN, or SAN NAALADase: 59 AA's downstream
from STQK (SEQ ID NO: 3) motif: SFG Site III: PBP: 111 AA's
downstream from SGC motif: KTG NAALADase: 110 AA's downstream from
SFG motif: KLG Site IV: PBP: 41 AA's downstream from SGC motif:
ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from SFG motif:
ERGV (SEQ ID NO: 4)
[0017] Since the beta-lactams provide their inhibition of PBP
transpeptidation of bacterial cell wall by binding to these four
active sites, one can infer that the conserved similarity in active
site sequences and location would confer similar binding properties
of moxalactam and other .beta.-lactam compounds to NAALADase and
possibly other neurogenic enzymes having sequences overlapping with
the four active binding site motif. Recent computer modeling
experiments have shown that while clavulanic acid exhibits a good
fit with NAALADase, moxalactam does not, suggesting another
neurogenic target for moxalactam. Further molecular modeling
studies have suggested that the neurogenic target for moxalactam is
another neurogenic peptidase, carboxypeptidase E. That discovery
coupled with observation of the significant behavioral modification
effects deriving from administration of very low doses of certain
penicillin protein binding compounds has provided insight into a
novel approach to the prevention and treatment of disease states
characterized by neurological dysfunction.
[0018] The unique neurological activity profiles of the two
.beta.-lactam compounds that have been studied most extensively to
date, moxalactam and clavulanic acid, suggest that those compounds
exhibit activity on multiple neurogenic enzyme targets, including
NAALADase and structurally related enzymes, particularly those that
might share the four active binding site motif common to both PBP
and NAALADase. To identify other putative neurogenic targets for
the behavioral and cognitive activities discovered for moxalactam
and clavulanic acid, the sequence for NAALADase II was used to
search the human genome database (NCBI-BLAST). Seven human gene
sequences were identified that have significant homology with
NAALADase H and that encode for the four active site motif:
TABLE-US-00003 1) >dbj/AP001769.2/AP001769 Homo sapiens
chromosome 11 clone RP11-240F8 map 11q14 Site I: PBP: 35 AA's
downstream from N-terminus . . . STTK (SEQ ID NO: 1) NAALADase: 38
AA's downstream from N-terminus: STQK (SEQ ID NO: 3)
>dbj/AP001769: NSRK (SEQ ID NO: 5) Site II: PBP: 57 AA's
downstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, or SAN
NAALADase: 59 AA's downstream from STQK (SEQ ID NO:3) motif: SFG
>dbj/AP001769: SFG Site III PBP: 111 AA's downstream from SGC
motif . . . KTG NAALADase: 110 AA's downstream from SFG motif: KLG
>dbj/AP001769: KLG Site IV: PBP: 41 AA's downstream from SGC
motif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream from
SFG motif: ERGV (SEQ ID NO: 4) >dbj/AP001769: ERSI (SEQ ID NO:
6) 2) >dbj|AP000827.2|AP000827 Homo sapiens chromosome 11 clone
RP. Site I: PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ
ID NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ
ID NO: 3) >dbj|AP000827.2: NSRK (SEQ ID NO: 5) Site II: PBP: 57
AA's downstream from STTK (SEQ ID NO: 1) motif: . . . SGC, SGN, or
SAN NAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif:
SFG >dbj|AP000827.2: SFG Site III: PBP:111 AA's downstream from
SGC motif . . . KTG NAALADase: 110 AA's downstream from SFG motif:
KLG >dbj|AP000827.2: KLG Site IV: PBP: 41 AA's downstream from
SGC motif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's downstream
from SFG motif: ERGV (SEQ ID NO: 4) >dbj|AP000827.2: ERSI (SEQ
ID NO: 6) 3) >dbj|AP000648.2|AP000648 Homo sapiens chromosome 11
clone CM. Site I: PBP: 35 AA's downstream from N-terminus . . .
STTK (SEQ ID NO: 1) NAALADase: 38 AA's downstream from N-terminus:
STQK (SEQ ID NO: 3) >>dbj.andgate.AP000648.2: NSRK (SEQ ID
NO: 5) Site II: PBP: 57 AA's downstream from STTK (SEQ ID NO: 1)
motif . . . SGC, SGN, or SAN NAALADase: 59 AA's downstream from
STQK (SEQ ID NO: 3) motif: SFG >dbj|AP000648.2: SFG Site III
PBP:111 AA's downstream from SGC motif . . . KTG NAALADase: 110
AA's downstream from SFG motif: KLG >dbj|AP000648.2: KLG Site
IV: PBP: 41 AA's downstream from SGC motif . . . ENKD (SEQ ID NO:
2) NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ ID NO:
4) >dbj|AP000648.2: ERSI (SEQ ID NO: 6) 4)
>gb|AC074003.2|AC074003 Homo sapiens chromosome 2 clone RP11.
Site I: PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID
NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID
NO: 3) gb|AC074003.2|AC074003: STQ- Site II: PBP: 57 AA's
downstream from STTK (SEQ ID NO: 1) motif: . . . SGC, SGN, or SAN
NAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif: SFG
gb|AC074003.2|AC074003: SFG Site III: PBP: 111 AA's downstream from
SGC motif . . . KTG NAALADase: 110 AA's downstream from SFG motif:
KLG gb|AC074003.2|AC074003: KLG Site IV: PBP: 41 AA's downstream
from SGC motif . . . ENKD (SEQ ID NO: 2) NAALADase: 41 AA's
downstream from SFG motif: ERGV (SEQ ID NO: 4)
gb|AC074003.2|AC074003 ERGV (SEQ ID NO: 4) 5)
>emb|AL162372.6|AL162372 Homo sapiens chromosome 13 clone RP.
Site I: PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID
NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID
NO: 3) emb|AL162372.6: STQ- Site II: PBP: 57 AA's downstream from
STTK (SEQ ID NO: 1) motif . . . SGC, SGN, or SAN NAALADase: 59 AA's
downstream from STQK (SEQ ID NO: 3) motif: SFG emb|AL162372.6: SFG
Site II: PBP: 111 AA's downstream from SGC motif . . . KTG
NAALADase: 110 AA's downstream from SFG motif: KLG emb|AL162372.6:
KLG Site IV: PBP: 41 AA's downstream from SGC motif . . . ENKD (SEQ
ID NO: 2) NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ
ID NO: 4) emb|AL162372.6
ERGV (SEQ ID NO: 4) 6) gb|AC024234.5|AC024234 Homo sapiens
chromosome 11 clone RP1. Site I: PBP: 35 AA's downstream from
N-terminus . . . STTK (SEQ ID NO: 1) NAALADase: 38 AA's downstream
from N-terminus: STQK (SEQ ID NO: 3) gb|AC024234.5|AC024234: STQ-
Site II: PBP: 57 AA's downstream from STTK (SEQ ID NO: 1) motif: .
. . SGC, SGN, or SAN NAALADase: 59 AA's downstream from STQK (SEQ
ID NO: 3) motif: SFG gb|AC024234.5|AC024234: SFG Site III: PBP: 111
AA's downstream from SGC motif . . . KTG NAALADase: 110 AA's
downstream from SFG motif KLG gb|AC024234.5|AC024234: KLG Site IV:
PBP: 41 AA's downstream from SGC motif . . . ENKD (SEQ ID NO: 2)
NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ ID NO: 4)
gb|AC024234.5|AC024234 ERGV (SEQ ID NO: 4) 7)
dbj|AP002369.1|AP002369 Homo sapiens chromosome 11 clone RP . . .
Site I: PBP: 35 AA's downstream from N-terminus . . . STTK (SEQ ID
NO: 1) NAALADase: 38 AA's downstream from N-terminus: STQK (SEQ ID
NO: 3) dbj|AP002369.1: STQ- Site II: PBP: 57 AA's downstream from
STTK (SEQ ID NO: 1) motif: . . . SGC, SGN, or SAN NAALADase: 59
AA's downstream from STQK (SEQ ID NO: 3) motif: SFG dbj|AP002369.1:
SFG Site III: PBP: 111 AA's downstream from SGC motif . . . KTG
NAALADase: 110 AA's downstream from SFG motif: KLG dbj|AP002369.1:
KLG Site IV: PBP: 41 AA's downstream from SGC motif . . . ENKD (SEQ
ID NO: 2) NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ
ID NO: 4) dbj|AP002369.1 ERGV (SEQ ID NO: 4)
[0019] The encoded protein of each of those gene sequences
expressed in the brain are probable targets for behavioral and
cognitive activity by .beta.-lactams and other NAALADase
inhibitors. Thus in accordance with one aspect of this invention
there is provided a method for modifying behavior and/or cognition
comprising the step of inhibiting the biological activity of the
non-NAALADase protein(s) expressed by one or more of the
above-identified gene sequences, by administering an effective
amount of a .beta.-lactam compound or other compound capable of
peptidase inhibition. As stated above, recent molecular modeling
studies now suggest that carboxypeptidase E is the enzyme which is
inhibited by moxalactam in neural tissues to provide basis for a
multiplicity of neurotherapeutic effects.
[0020] In one embodiment the peptidase inhibitors effective for use
in the various pharmaceutical formulation and method embodiments of
this invention, generally speaking, are compounds which exhibit
detectable selective affinity for art recognized penicillin-binding
proteins, including particularly .beta.-lactam-containing compounds
(hereinafter ".beta.-lactam compounds") such as penicillins and
cephalosporins, and most preferably, ceratin 1-oxa-1-dithiacephem
analogues thereof, certain .beta.-lactamase inhibitors, and
peptides comprising the amino acid sequence
Ala-D-.gamma.-Glu-Lys-D-Ala-D-Ala. Among such peptidase inhibiting
compounds, those preferred for use in accordance with this
invention are compounds that also exhibit good blood brain barrier
transport properties evidenced by favorable cerebral spinal fluid
(CSF)/brain:serum concentration ratios. Further, it will be
appreciated that other art-recognized peptidase inhibitors may be
used alone or in combination with penicillin protein-binding
compounds for treatment and prevention of behavioral and/or
cognitive disorders.
[0021] In the embodiments of the invention directed to
pharmaceutical formulations for use in inhibition of neurogenic
peptidase to modify behavior and/or improve cognitive function, the
.beta.-lactam compounds are typically formulated in unit dosage
form optionally in combination with, or as covalent conjugates of,
other compounds or molecular entities, respectively, known to
enhance drug transport across the blood brain barrier. Such drug
formulation/conjugation techniques are described and claimed in the
following listed United States Patents: U.S. Pat. Nos. 5,624,894;
5,672,683; 5,525,727; 5,413,996; 5,296,483; 5,187,158; 5,177,064;
5,082,853; 5,008,257; 4,933,438; 4,900,837; 4,880,921; 4,824,850;
4,771,059; and 4,540,564.
[0022] Enhanced concentrations of drug substances in the brain can
also be achieved by co-administration with P-glycoprotein efflux
inhibitors such as those described in U.S. Pat. Nos. 5,889,007;
5,874,434; 5,654,304; 5,620,855; 5,643,909; and 5,591,715.
Alternatively, .beta.-lactam antibiotic compounds useful in
accordance with this invention, including particularly
1-oxa-1-dethia cephems, can be administered alone or in combination
with art-recognized .beta.-lactamase inhibitors, which themselves
may or may not be .beta.-lactam compounds or compounds capable of
exhibiting selective affinity for penicillin-binding proteins.
Examples of .beta.-lactamase inhibitors which can be used in
combination with other neuropeptidase inhibitors useful in
accordance with this invention for treatment and/or prevention of
cognitive or behavioral disorders are other .beta.-lactam compounds
which may or may not exhibit independent clinically significant
antibacterial activity, such as clavulanic acid and thienamycin and
analogs thereof, sulbactam, tazobactam, sultamicillin, and
aztreonam and other monolactams.
[0023] The patent and non-patent literature is replete with
references describing .beta.-lactam antibiotics, their preparation,
their characterization, their formulation and their mode of action.
.beta.-Lactam antibiotics are known to exhibit their antibiotic
activity by interfering with one or more biological pathways
involved in bacteria cell wall synthesis; more particularly, they
inhibit carboxypeptidase and/or transpeptidase (or protease)
activity involved in cross-linking of the peptidoglycan chains used
as building blocks for cell wall synthesis. .beta.-Lactam
antibiotics are thus believed to act as inhibitors of
carboxypeptidases or transpeptidases by their covalent, and by some
reports, noncovalent associative bonding, to one or more of a group
of such bacterial enzymes generally termed penicillin binding
proteins (PBP's). Such enzymes serve to complete bacteria cell wall
synthesis by cross linking peptidoglycan chains.
[0024] A similar peptidase-substrate interaction/inhibition is now
suggested in accordance with this invention as a significant
neurochemical pathway involved in brain function pivotal to
cognitive performance and behavioral phenotype. Such a
neurochemical mechanism is suggested too by the discovery that
delivery of effective amounts of the peptide
Ala-D-.gamma.-Glu-Lys-D-alanyl-D-alanine directly into the brain
produced the same modified behavioral characteristics as that
achieved by comparable concentrations of .beta.-lactam compounds in
the brain. The peptide appears to serve as a substitute substrate
for (and thus serve to inhibit the activity thereof) one or more
neurogenic peptidases (e.g., NAALADases) that normally exhibit
their activity on peptidic neurotransmitters or neuromodulators,
i.e., NAAD, in the ordinary course of certain neurochemical
processes that mediate cognitive performance and behavioral
phenotype.
[0025] Based on animal tests to date it is believed that the
general classes of behavioral disorders can be prevented or treated
in accordance with this invention by administration of effective
amounts of inhibitors of other neurogenic peptidases, include
aggressive disorder, obsessive-compulsive disorder, anxiety,
depression, and attention deficient hyperactivity disease (ADHD).
Thus in one embodiment of the invention a carboxypeptidase
inhibitor, more specifically an inhibitor of carboxypeptidase E, is
administered as an anti-aggressive agent to control impulsivity and
violence in a patient afflicted with autism, Tourette's Syndrome,
mental retardation, psychosis, mania, senile dementia or that in a
patient with personality disorder and history of inappropriate
aggression.
[0026] Other neurological disease states which can be treated in
accordance with the present invention include depression, including
major depression (single episode, recurrent, melancholic),
atypical, dysthmia, subsyndromal, agitated, retarded, co-morbid
with cancer, diabetes, or post-myocardial infarction, involutional,
bipolar disorder, psychotic depression, endogenous and reactive,
obsessive-compulsive disorder, or bulimia. In addition, peptidase
inhibitors can be used to treat patients suffering from pain (given
alone or in combination with morphine, codeine, or
dextroproposyphene), obsessive-compulsive personality disorder,
post-traumatic stress disorder, hypertension, atherosclerosis,
anxiety, anorexia nervosa, panic, social phobia, stuttering, sleep
disorders, chronic fatigue, cognition deficit associated with
Alzheimer's disease, alcohol abuse, appetite disorders, weight
loss, agoraphobia, improving memory, amnesia, smoking cessation,
nicotine withdrawal syndrome symptoms, disturbances of mood and/or
appetite associated with pre-menstrual syndrome, depressed mood
and/or carbohydrate craving associated with pre-menstrual syndrome,
disturbances of mood, disturbances of appetite or disturbances
which contribute to recidivism associated with nicotine withdrawal,
circadian rhythm disorder, borderline personality disorder,
hypochondriasis, pre-menstrual syndrome (PMS), late luteal phase
dysphoric disorder, pre-menstrual dysphoric disorder,
trichotillomania, symptoms following discontinuation of other
antidepressants, aggressive/intermittent explosive disorder,
compulsive gambling, compulsive spending, compulsive sex,
psychoactive substance use disorder, sexual disorder,
schizophrenia, premature ejaculation, or psychiatric symptoms
selected from stress, worry, anger, rejection sensitivity, and lack
of mental or physical energy.
[0027] Other examples of pathologic, psychologic conditions which
may be treated in accordance with this invention include, but are
not limited to: Moderate Mental Retardation (318.00), Severe Mental
Retardation (318.10), Profound Mental Retardation (318.20),
Unspecified Mental Retardation (319.00), Autistic Disorder
(299.00), Pervasive Development Disorder NOS (299.80),
Attention-Deficit Hyperactivity Disorder (314.01), Conduct
Disorder, Group Type (312.20), Conduct Disorder, Solitary
Aggressive Type (312.00), Conduct Disorder, Undifferentiated Type
(312.90), Tourette's Disorder (307.23), Chronic Motor or Vocal Tic
Disorder (307.22), Transient Tic Disorder (307.21), Tic Disorder
NOS (307.20), Primary Degenerative Dementia of the Alzheimer Type,
Senile Onset, Uncomplicated (290.00), Primary Degenerative Dementia
of The Alzheimer Type, Senile Onset, with Delirium (290.30),
Primary Degenerative Dementia of the Alzheimer Type, Senile Onset,
with Delusions (390.20), Primary Degenerative Dementia of the
Alzheimer Type, Senile Onset, with Depression (290.21), Primary
Degenerative Dementia of the Alzheimer Type, Presenile Onset,
Uncomplicated (290.10), Primary Degenerative Dementia of the
Alzheimer Type, Presenile Onset, with Delirium (290.11), Primary
Degenerative Dementia of the Alzheimer Type, Presenile Onset, with
Delusions (290.12), Primary Degenerative Dementia of the Alzheimer
Type, Presenile Onset, with Depression (290.13), Multi-infarct
dementia, Uncomplicated (290.40), Multi-infarct dementia, with
Delirium (290.41), Multi-infarct Dementia, with Delusions (290.42),
Multi-infarct Dementia, with Depression (290.4 3), Senile Dementia
NOS (290.10), Presenile Dementia NOS (290.10), Alcohol Withdrawal
Delirium (291.00), Alcohol Hallucinosis (291.30), Alcohol Dementia
Associated with Alcoholism (291.20), Amphetamine or Similarly
Acting Sympathomimetic Intoxication (305.70), Amphetamine or
Similarly Acting Sympathomimetic Delusional Disorder (292.11),
Cannabis Delusional Disorder (292.11), Cocaine Intoxication
(305.60), Cocaine Delirium (292.81), Cocaine Delusional Disorder
(292.11), Hallucinogen Hallucinosis (305.30), Hallucinogen
Delusional Disorder (292.11), Hallucinogen Mood Disorder (292.84),
Hallucinogen Posthallucinogen Perception Disorder (292.89),
Phencyclidine (PCP or Similarly Acting Arylcyclohexylamine
Intoxication (305.90), Phencyclidine (PCP) or Similarly Acting
Arylcyclohexylamine Delirium (292.81), Phencyclidine (PCP) or
Similarly Acting Arylcyclohexylamine Delusional Disorder (292.11),
Phencyclidine (PCP) or Similarly Acting Arylcyclohexylamine Hood
Disorder (292.84), Phencyclidine (PCP) or Similarly Acting
Arylcyclohexylamine Organic Mental Disorder NOS (292.90), Other or
unspecified Psychoactive Substance Intoxication (305.90), Other or
Unspecified Psychoactive Substance Delirium (292.81), Other or
Unspecified Psychoactive Substance Dementia (292.82), Other or
Unspecified Psychoactive Substance Delusional Disorder (292.11),
Other or Unspecified Psychoactive Substance Hallucinosis (292.12),
Other or Unspecified Psychoactive Substance Mood Disorder (292.84),
Other or Unspecified Psychoactive Substance Anxiety Disorder
(292.89), Other or Unspecified Psychoactive Substance Personality
Disorder (292.89), Other or Unspecified Psychoactive Substance
Organic Mental Disorder NOS (292.90), Delirium (293.00), Dementia
(294.10), Organic Delusional Disorder (293.81), Organic
Hallucinosis (293.81), Organic Mood Disorder (293.83), Organic
Anxiety Disorder (294.80), Organic Personality Disorder (310.10),
Organic Mental Disorder (29.80), Obsessive Compulsive Disorder
(300.30), Post-traumatic Stress Disorder (309.89), Generalized
Anxiety Disorder (300.02), Anxiety Disorder NOS (300.00), Body
Dysmorphic Disorder (300.70), Hypochondriasis (or Hypochondriacal
Neurosis) (300.70), Somatization Disorder (300.81),
Undifferentiated Somatofoiin Disorder (300.70), Somatoform Disorder
NOS (300.70), Intermittent Explosive Disorder (312.34), Kleptomania
(312.32), Pathological Gambling (312.31), Pyromania (312.33),
Trichotillomania (312.39), and Impulse Control Disorder NOS
(312.39).
[0028] Additional examples of pathologic psychological conditions
which may be treated using .beta.-lactam containing peptidase
inhibitors as described in this invention include Schizophrenia,
Catatonic, Subchronic, (295.21), Schizophrenia, Catatonic, Chronic
(295.22), Schizophrenia, Catatonic, Subchronic with Acute
Exacerbation (295.23), Schizophrenia, Catatonic, Chronic with Acute
Exacerbation (295.24), Schizophrenia, Catatonic, in Remission
(295.55), Schizophrenia, Catatonic, Unspecified (295.20),
Schizophrenia, Disorganized, Chronic (295.12), Schizophrenia,
Disorganized, Subchronic with Acute Exacerbation (29 5.13),
Schizophrenia, Disorganized, Chronic with Acute Exacerbation
(295.14), Schizophrenia, Disorganized, in Remission (295.15),
Schizophrenia, Disorganized, Unspecified (295.10), Schizophrenia,
Paranoid, Subchronic 295.31), Schizophrenia, Paranoid, Chronic
(295.32), Schizophrenia, Paranoid, Subchronic with Acute
Exacerbation (295.33), Schizophrenia, Paranoid, Chronic with Acute
Exacerbation (295.34), Schizophrenia, Paranoid, in Remission
(295.35), Schizophrenia, Paranoid, Unspecified (295.30),
Schizophrenia, Undifferentiated, Subchronic (295.91),
Schizophrenia, Undifferentiated, Chronic (295.92), Schizophrenia,
Undifferentiated, Subchronic with Acute Exacerbation (295.93),
Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation
(295.94), Schizophrenia, Undifferentiated, in Remission (295.95),
Schizophrenia, Undifferentiated, Unspecified (295.90),
Schizophrenia, Residual, Subchronic (295.61), Schizophrenia,
Residual, Chronic (295.62), Schizophrenia, Residual, Subchronic
with Acute Exacerbation (295.63), Schizophrenia, Residual, Chronic
with Acute Exacerbation (295.94), Schizophrenia, Residual, in
Remission (295.65), Schizophrenia, Residual, unspecified (295.60),
Delusional (Paranoid) Disorder (297.10), Brief Reactive Psychosis
(298.80), Schizophreniform Disorder (295.40), Schizoaffective
Disorder (295.70), induced Psychotic Disorder (297.30), Psychotic
Disorder NOS (Atypical Psychosis) (298.90), Bipolar Disorder,
Mixed, Severe, without Psychotic Features (296.63), Bipolar
Disorder, Manic, Severe, without Psychotic Features (296.43),
Bipolar Disorder, Depressed, Severe, without Psychotic Features
(296.53), Bipolar Disorder, Mixed, with Psychotic Features
(296.64), Bipolar Disorder, Manic, with Psychotic Features
(296.44), Bipolar Disorder, Depressed, with Psychotic Features
(296.54), Bipolar Disorder NOS (296.70), Major Depression, Single
Episode, with Psychotic Features (296.24), Major Depression,
Recurrent with Psychotic Features (296.34) Personality Disorders,
Paranoid (301.00), Personality Disorders, Schizoid (301.20),
Personality Disorders, Schizotypal (301.22), Personality Disorders,
Antisocial (301.70), Personality Disorders, Borderline
(301.83).
[0029] Anxiety disorders which may be treated in accordance with
this invention include, but are not limited to, Anxiety Disorders
(235), Panic Disorder (235), Panic Disorder with Agoraphobia
(300.21), Panic Disorder without Agoraphobia (300.01), Agoraphobia
without History of Panic Disorders (300.22), Social Phobia
(300.23), Simple Phobia (300.29), Organic Anxiety Disorder
(294.80), Psychoactive Substance Anxiety Disorder (292.89),
Separation Anxiety Disorder (309.21), Avoidant Disorder of
Childhood or Adolescence (313.21), and Overanxious Disorder
(313.00).
[0030] Effective amounts of the .beta.-lactam carboxypeptidase
inhibiting compounds described herein, can be used for the
treatment of the following pathologic psychological conditions:
Moderate Mental Retardation; Severe Mental Retardation; Profound
Mental Retardation; Autistic Disorder; Attention-Deficit
Hyperactivity Disorder; Pervasive Development Disorder NOS; Conduct
Disorder, Group Type; Conduct Disorder, Solitary Aggressive Type;
Tourette's Disorder; Primary Degenerative Dementia of the Alzheimer
Type, Senile Onset, with Delirium; Primary Degenerative Dementia of
the Alzheimer Type, Senile Onset, with Delusions; Primary
Degenerative Dementia of the Alzheimer Type, Presenile Onset;
Schizophrenia, Catatonic, Subchronic; Schizophrenia, Catatonic,
Chronic; Schizophrenia, Catatonic, Subchronic with Acute
Exacerbation; Schizophrenia, Catatonic, Chronic with Acute
Exacerbation; Schizophrenia, Catatonic, in Remission;
Schizophrenia, Catatonic, Unspecified; Schizophrenia, Disorganized,
Subchronic; Schizophrenia, Disorganized, Chronic; Schizophrenia,
Disorganized, Subchronic with Acute Exacerbation; Schizophrenia,
Disorganized, Chronic with Acute Exacerbation; Schizophrenia,
Disorganized, in Remission; Schizophrenia, Disorganized,
Unspecified; Schizophrenia, Paranoid, Subchronic; Schizophrenia,
Paranoid, Chronic; Schizophrenia, Paranoid, Subchronic with Acute
Exacerbation; Schizophrenia, Paranoid, Chronic with Acute
Exacerbation; Schizophrenia, Paranoid, in Remission; Schizophrenia,
Paranoid, Unspecified; Schizophrenia, Undifferentiated, Subchronic;
Schizophrenia, Undifferentiated, Chronic; Schizophrenia,
Undifferentiated, Subchronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, in Remission; Schizophrenia,
Undifferentiated, Unspecified; Schizophrenia, Residual, Subchronic;
Schizophrenia, Residual Chronic; Schizophrenia, Residual,
Subchronic with Acute Exacerbation; Schizophrenia, Residual,
Chronic with Acute Exacerbation; Schizophrenia, Residual, in
Remission; Schizophrenia, Residual, Unspecified; Delusional
(Paranoid) Disorder; Brief Reactive Psychosis; Schizophreniform
Disorder; Schizoaffective Disorder; Induced Psychotic Disorder;
Psychotic Disorder NOS (Atypical Psychosis); Bipolar Disorder,
Mixed, with Psychotic Features; Bipolar Disorder, Manic, with
Psychotic Features; Bipolar Disorder, Depressed, with Psychotic
Features; Bipolar Disorder NOS; Major Depression, Single Episode,
or Recurrent with Psychotic Features; Personality Disorders,
Paranoid; Personality Disorders, Schizoid; Personality Disorders,
Schizotypal; Personality Disorders, Antisocial; Personality
Disorders, Borderline, Anxiety Disorders, Panic Disorder, Panic
Disorder with Agoraphobia, Panic Disorder without Agoraphobia,
Agoraphobia without History of Panic Disorders, Social Phobia,
Simple Phobia, Obsessive Compulsive Disorder, Post-Traumatic Stress
Disorder, Generalized Anxiety Disorder, Anxiety Disorder NOS,
Organic Anxiety Disorder, Psychoactive Substance Anxiety Disorder,
Separation Anxiety Disorder, Avoidant Disorder of Childhood or
Adolescence, and Overanxious Disorder.
[0031] One or more inhibitors of neurogenic NAALADase, including
particularly neurotropic .beta.-lactam antibiotics exhibiting
carboxypeptidase E inhibition activity, or .beta.-lactamase
inhibitors can be used alone, in combination or in combination with
P-glycoprotein inhibitors to treat the following psychotic
conditions: Schizophrenia, Catatonic, Subchronic; Schizophrenia,
Catatonic, Chronic; Schizophrenia, Catatonic, Subchronic with Acute
Exacerbation; Schizophrenia, Catatonic, Chronic with Acute
Exacerbation; Schizophrenia, Catatonic, in Remission;
Schizophrenia, Catatonic, Unspecified; Schizophrenia, Disorganized,
Subchronic; Schizophrenia, Disorganized, Chronic; Schizophrenia,
Disorganized, Subchronic with Acute Exacerbation; Schizophrenia,
Disorganized, Chronic with Acute Exacerbation; Schizophrenia,
Disorganized, in Remission; Schizophrenia, Disorganized,
Unspecified; Schizophrenia, Paranoid, Subchronic; Schizophrenia,
Paranoid, Chronic; Schizophrenia, Paranoid, Subchronic with Acute
Exacerbation; Schizophrenia, Paranoid, Chronic with Acute
Exacerbation; Schizophrenia, Paranoid, in Remission; Schizophrenia,
Paranoid, Unspecified; Schizophrenia, Undifferentiated, Subchronic;
Schizophrenia, Undifferentiated, Chronic; Schizophrenia,
Undifferentiated, Subchronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, in Remission; Schizophrenia,
Undifferentiated, Unspecified; Schizophrenia, Residual, Subchronic;
Schizophrenia, Residual, Chronic; Schizophrenia, Residual,
Subchronic with Acute Exacerbation; Schizophrenia, Residual,
Chronic with Acute Exacerbation; Schizophrenia, Residual, in
Remission; Schizophrenia, Residual, Unspecified; Delusional
(Paranoid) Disorder; Brief Reactive Psychosis; Schizophreniform
Disorder; Schizoaffective Disorder; Induced Psychotic Disorder;
Psychotic Disorder NOS (Atypical Psychosis); Bipolar Disorder,
Mixed, with Psychotic Features; Bipolar Disorder, Manic, with
Psychotic Features; Bipolar Disorder, Depressed, with Psychotic
Features; Bipolar Disorder NOS; Personality Disorders, Paranoid;
Personality Disorders, Schizoid; Personality Disorders,
Schizotypal; Personality Disorders, Antisocial; Personality
Disorders, Borderline.
[0032] Examples of psychotic conditions which are most preferredly
treated in accordance with the method of this invention include
Schizophrenia, Catatonic, Subchronic; Schizophrenia, Catatonic,
Chronic; Schizophrenia, Catatonic, Subchronic with Acute
Exacerbation; Schizophrenia, Catatonic, Chronic with Acute
Exacerbation; Schizophrenia, Catatonic, in Remission;
Schizophrenia, Catatonic, Unspecified; Schizophrenia, Disorganized,
Subchornic; Schizophrenia, Disorganized, Chronic; Schizophrenia,
Disorganized, Subchronic with Acute Exacerbation; Schizophrenia,
Disorganized, Chronic with Acute Exacerbation; Schizophrenia,
Disorganized, in Remission; Schizophrenia, Disorganized,
Unspecified; Schizophrenia, Paranoid, Subchronic; Schizophrenia,
Paranoid, Chronic; Schizophrenia, Paranoid, Subchronic with Acute
Exacerbation; Schizophrenia, Paranoid, Chronic with Acute
Exacerbation; Schizophrenia, Paranoid, in Remission; Schizophrenia,
Paranoid, Unspecified; Schizophrenia, Undifferentiated, Subchronic;
Schizophrenia, Undifferentiated, Chronic; Schizophrenia,
Undifferentiated, Subchronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation;
Schizophrenia, Undifferentiated, in Remission; Schizophrenia,
Undifferentiated, Unspecified; Schizophrenia, Residual, Subchronic;
Schizophrenia, Residual, Chronic; Schizophrenia, Residual,
Subchronic with Acute Exacerbation; Schizophrenia, Residual,
Chronic with Acute Exacerbation; Schizophrenia, Residual, in
Remission; Schizophrenia, Residual, Unspecified; Delusional
(Paranoid) Disorder; Brief Reactive Psychosis; Schizophreniform
Disorder; Schizoaffective Disorder; Personality Disorders,
Schizoid; and Personality Disorders, Schizotypal.
[0033] In one preferred aspect of this invention there is provided
a treatment for anxiety. Examples of anxiety disorders which are
treated using the present method and pharmaceutical formulations of
this invention, include Anxiety Disorders, Panic Disorder, Panic
Disorder with Agoraphobia, Panic Disorder without Agoraphobia,
Agoraphobia without History of Panic Disorders, Social Phobia,
Simple Phobia, Obsessive Compulsive Disorder, Post-Traumatic Stress
Disorder, Generalized Anxiety Disorder, Anxiety Disorder NOS,
Organic Anxiety Disorder, Psychoactive Substance Anxiety Disorder,
Separation Anxiety Disorder, Avoidant Disorder of Childhood or
Adolescence, and Overanxious Disorder.
[0034] Examples of the anxiety disorders which are most preferredly
treated include Panic Disorder; Social Phobia; Simple Phobia;
Organic Anxiety Disorder; Obsessive Compulsive Disorder;
Post-traumatic Stress Disorder; Generalized Anxiety Disorder; and
Anxiety Disorder NOS.
[0035] The compounds used as the neurochemically functional agent
in the methods and formulations of the present invention are, in
one embodiment of the invention, characterized particularly by
their binding to penicillin-binding proteins [as determined using
methods described, for example, by B. G. Spratt, Properties of the
penicillin-binding proteins of Escherichia coli K12, Eur. J.
Biochem., 72:341-352 (1977) and N. H. Georgopapadakou, S. A. Smith,
C. M. Cimarusti, and R. B. Sykes, Binding of monolactams to
penicillin-binding proteins of Escherichia coli and Staphylococcus
aureus: Relation to antibacterial activity, Antimocrob. Agents
Chemother., 23:98-104 (1983)] and, in the case of antibiotics, by
their inhibition of selective carboxypeptidase and/or
transpeptidase activity on peptide substrates comprising the amino
acid sequence Ala-D-.gamma.-Glu-Lys-D-alanyl-D-alanine. Such
compounds include particularly, .beta.-lactam compounds, including
penicillins, cephalosporins, and monocyclic and bicyclic analogs
and/or derivatives thereof. Commercially available antibiotics for
use in the methods and manufacture of pharmaceutical formulations
of this invention include penams, cephems, 1-oxa-1-dethia cephems,
clavams, clavems, azetidinones, carbapenems, carbapenems and
carbacephems.
[0036] In one preferred embodiment of the present invention the
peptidase inhibitor is a compound of the formula:
##STR00001##
wherein R is hydrogen, a salt forming group or an active ester
forming group; R.sup.1 is hydrogen or C.sub.1-C.sub.4 alkoxy; X is
O, S.dbd.O, SO.sub.2, or C; T is C.sub.1-C.sub.4 alkyl, halo
(including chloro, fluoro, bromo and iodo), hydroxy,
O(C.sub.1-C.sub.4)alkyl, or --CH.sub.2B wherein B is the residue of
a nucleophile B:H, and Acyl is the residue of an organic acid Acyl
OH.
[0037] Examples of such commercially available compounds
(1-alkoxy-1-dethia cephems) are moxalactam and flomoxef. Moxalactam
is described and claimed in U.S. Pat. No. 4,323,567. Moxalactam is
particularly preferred due to its good blood-brain barrier
transport thus providing a relatively high concentration ratio of
that compound in the brain relative to blood/serum levels.
[0038] In another embodiment invention moxalactam or another
commercially available .beta.-lactam antibiotic (or derivative or
analogue thereof) detailed for parenteral administration to achieve
clinically effective antibiotic tissue concentrations, is converted
to the corresponding mono- or bis-active esters to improve oral
absorption of said compounds to a level sufficient to inhibit
neurogenic peptidase activity in the brain and concomitantly effect
behavior and cognitive performance, albeit at a serum concentration
insufficient for clinical antibiotic efficacy.
[0039] Examples of suitable in vivo hydrolysable (active) ester
groups include, for example, acyloxyalkyl groups such as
acetoxymethyl, pivaloyloxymethyl, .beta.-acetoxyethyl,
.beta.-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy) prop-1-yl, and
(1-aminoethyl) carbonyloxymethyl; alkoxycarbonyloxyalkyl groups,
such as ethoxycarbonyloxymethyl and alpha-ethoxycarbonyloxyethyl;
dialkylaminoalkyl groups, such as ethoxycarbonyloxymethyl and
(3-ethoxycarbonyloxyethyl; dialkylaminoalkyl especially di-lower
alkylamino alkyl groups such as dimethylaminomethyl,
dimethylaminoethyl, diethylaminomethyl or
diethylaminoethyl:2-(alkoxycarbonyl)-2-alkenyl groups such as
2-(isobutoxycarbonyl) pent-2-enyl and 2-(ethoxycarbonyl)but-2-enyl;
lactone groups such as phthalidyl and dimethoxyphthalidyl; and
esters linked to a second .beta.-lactam antibiotic or to a
.beta.-lactamase inhibitor. One example of such chemical
modification of a commercially available parenteral .beta.-lactam
antibiotic is moxalactam (Ia, Y.dbd.OH, R.sub.1.dbd.OCH.sub.3, and
V.dbd.COM wherein, M=OH) is the preparation one of its active ester
analogue Ia wherein Y.dbd.OM, M=H or an active ester, e.g.,
1-indanyl and V.dbd.CO.sub.2M wherein M is H or an active ester and
wherein at least one of V and Y include an active ester moiety.
[0040] Suitable pharmaceutically acceptable salts of the carboxy
group of the above identified .beta.-lactam antibiotics include
metal salts, e.g. aluminum, alkali metal salts such as sodium or
potassium, alkaline earth metal salts such as calcium or magnesium,
and ammonium or substituted ammonium salts, for example those with
lower alkylamines such as triethylamine, hydroxy-lower alkylamines
such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine or
tris-(2-hydroxyethyl)amine, cycloalkylamines such as
dicyclohexylamine, or with procaine, dibenzylamine,
N,N-dibenzylethylenediamine, 1-ephenamine, N-methylmorpholine,
N-ethylpiperidine, N-benzyl-.beta.-phenethylamine,
dehydroabietylamine, N.N'-bisdehydro-abietylamine, ethylenediamine,
or bases of to pyridine type such as pyridine, collidine or
quinoline, or other amines which have been used to form salts with
known penicillins and cephalosporins. Other useful salts include
the lithium salt and silver salt. Salts within compounds of formula
(I), may be prepared by salt exchange in conventional manner.
[0041] In another embodiment of the present invention a penicillin
or penicillin analog of the formula
##STR00002##
is employed wherein said formula X.dbd.O, S, SO, SO.sub.2 or C; R
is H or a pharmaceutical acceptable salt-forming or ester-forming
group; R.sup.1 is H or lower alkoxy, G is hydrogen or hydroxy, and
Z is amino, acylamino, CO.sub.2M, SO.sub.3M, PO.sub.3M.sub.2 or
PO.sub.2M wherein M is hydrogen or a pharmaceutically acceptable
salt-forming or ester-forming group, preferably an active
ester-forming group.
[0042] Non-antibiotic or weakly antibiotic penam and cephem or
cephem sulfoxides and sulfones and structurally related
.beta.-lactamase inhibitors such as tazobactam, clavulanic acid and
sulbactam, are particularly useful in applications where
development of antibiotic resistance is of concern.
[0043] Animal tests indicate a threshold effective dose of
moxalactam (administered parenterally) to be about 50 .mu.g/kg of
body weight. Based on animal test data and on the known
distribution of parenterally administered moxalactam between the
brain and other body tissues, that the effective minimum neurogenic
peptidase inhibiting, concentration of moxalactam in the brain is
about 30 nM. Clavulanic acid has been shown to be an effective
inhibitor of neurogenic NAALADase when administered i.p. at less
than 1 microgram per kilogram of body weight. The range of
effective dosage levels of the inhibitors when used in the
treatment of behavioral and/or cognitive disorders in accordance
with this invention will depend significantly on patient body
weight, the affinity of the inhibitor for the target neurogenic
protease, the blood-brain barrier transport characteristics of the
active compound, the mode of administration and the optional use of
available drug formulations/conjugation technologies available for
enhancement of blood-brain barrier transport. For parenterally
administered moxalactam the minimum effective dose in hamsters and
other test species is about 50 micrograms per kg of body weight,
more or less. The use of moxalactam in an oral dosage form,
preferably modified or derivatized in the form of an active ester,
is estimated to range from about 2.5 to about 50 mg per dose, much
less than the dose of moxalactam necessary to provide
therapeutically effective antibiotic concentration. The effective
oral dose of clavulanate is expected to be about 0.1 to about 10
mgs per dose. Clavulanate is orally absorbed and it exhibits good
blood brain barrier transport.
[0044] The effective doses of other peptidase inhibitors will vary,
again depending on their inherent affinity for the target
peptidase, the selected route of administration, patient weight,
and blood-brain barrier transport efficiency. The effective dosages
of peptidase inhibitors used in accordance with the present
invention can be readily determined empirically using animal models
coupled with use of art recognized analytical techniques.
Typically, the dosage levels for .beta.-lactam antibiotic compounds
used in the methods and formulations of this invention is less than
that necessary to achieve clinically effective antibacterial
levels. Parenteral dosages of .beta.-lactam antibiotic compounds
can range from about 1 to about 80 mg per dose. Oral dosages can
range from about 2.5 to about 150 mg per dose. Higher or lower
dosage amounts may be appropriate and used in accordance with this
invention when patient circumstances dictate such in the judgment
of the attending physician. Thus, for example, where
patient/clinical conditions are such that the inherent antibiotic
activity of the .beta.-lactam compounds are not considered to be a
complicating contraindication, higher doses of the antibiotic up to
or exceeding the dosage levels capable of providing threshold
clinically effective antibiotic blood levels can be used to treat
patients in need of therapy effected by peptidase inhibition in
accordance with this invention.
[0045] The present invention further provides certain
pharmaceutical formulations for treatment of behavioral or
cognitive disorders and other disease states associated with
production of abnormal glutamate concentrations in nervous tissues
and other tissues harboring NAALADase activity. Generally the
formulation comprises a neurologically active ingredient including
a compound capable of inhibiting a bacterial enzyme and capable of
inhibiting a neurogenic peptidase that is known, by empirical
evidence, to selectively act on a peptide comprising the amino acid
sequence Ala-D-.gamma.-Glu-Lys-D-alanyl-D-alanine, and a
pharmaceutically acceptable carrier therefor. In one embodiment the
pharmaceutical formulation in a unit dosage form comprises an
amount of a .beta.-lactam compound capable of inhibiting peptidase
activity in a patient experiencing or disposed to develop a
neurological condition that could be prevented or treated to reduce
its symptoms by peptidase inhibition. The amount of the peptidase
inhibitor and the nature of the carrier is dependent, of course, on
the intended route of administration. The amount of inhibitor is
that amount effective to provide upon delivery by the predetermined
route of administration, a concentration of the inhibitor in the
tissue where peptidase inhibition is desired, e.g., in the brain
effective to treat and reduce symptoms of the targeted behavioral
or cognitive disorders or other disorders than can be treated by
inhibition of peptidase activity. In embodiments utilizing
.beta.-lactam antibiotic compounds the amount of the peptidase
inhibitor in the present formulations is typically less than that
capable of providing clinically effective bacterial protease
inhibition, i.e., less than that capable of providing
antibiotically effective levels when administered to a patient in
the dosage form provided. The peptidase inhibitors for use in
accordance with this invention can be combined with one or more
pharmaceutically acceptable carriers, and may be administered, for
example, orally in such forms as tablets, capsules, caplets,
dispersible powders, granules, lozenges, mucosal patches, sachets,
and the like. The inhibitor can be combined with a pharmaceutically
acceptable carrier, for example starch, lactose or trehalose, alone
or in combination with one or more tableting excipients and pressed
into tablets or lozenges. Optionally, such tablets, caplets or
capsules can be enterically coated to minimize
hydrolysis/degradation in the stomach. Oral dosage formulations
contain about 1 to about 99% by weight active ingredient and about
1 to about 99% of a pharmaceutically acceptable carrier and/or
formulating excipients. Optionally, when .beta.-lactam antibiotics
are used as the inhibitors the dosage forms can be formulated by
combining it with a P-glycoprotein inhibitor to provide enhanced
drug half-life and brain concentrations of the active ingredient.
Alternatively, the protease inhibitor can simply be co-administered
with a P-glycoprotein or .beta.-lactamase inhibitor.
[0046] In another embodiment of the invention pharmaceutical
preparations may contain, for example, from about 2.5% to about 90%
of the active ingredient in combination with the carrier, more
usually between about 5% and about 60% by weight active ingredient.
The pharmaceutical formulations in accordance with one embodiment
of this invention are formulated for per os administration, i.e.,
oral ingestion administration or buccal or sublingual
administration (in the form of sachets, lozenges, and/or oral
mucosal patches). In another embodiment the dosage form is
formulated for per os administration in a prolonged release dosage
form formulated to release the active ingredient over a
predetermined period of time.
[0047] Topical dosage forms, including transdermal patches,
intranasal, and suppository dosage unit formulations containing the
active peptidase inhibitor and conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles
adapted for such routes of administration are also within the scope
of this invention.
[0048] The pharmaceutical formulations in accordance with this
invention alternatively can be delivered via parenteral routes of
administration, including subcutaneous administration,
intraperitoneal administration, intramuscular administration and
intravenous administration. Such parenteral dosage forms are
typically in the form of aqueous solutions or dispersions utilizing
a pharmaceutically acceptable carrier such as isotonic saline, 5%
glucose, or other well known pharmaceutically acceptable liquid
carrier composition.
[0049] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders or
lyophilizates for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the dosage form
must be sterile and it must be stable under the conditions of
manufacture and storage, and must be preserved against the
contaminating action of microorganisms. The carrier for injectable
formulations can be a solvent or dispersion medium containing, for
example, water, ethanol, or a polyol (or example glycerol,
propylene glycol and liquid polyethyleneglycol), mixtures thereof,
and vegetable oil.
[0050] Parenteral dosage forms of the peptidase inhibitors useful
for treatment of behavioral and cognitive disorders and other
disease states responsive to neurogenic peptidase inhibition can
also be formulated as injectable prolonged release formulations in
which the protease inhibitor is combined with one or more natural
or synthetic biodegradable or biodespersible polymers such as
carbohydrates, including starches, gums and etherified or
esterified cellulosic derivatives, polyethers, polyesters
(particularly polylactide, polygylcolide or
poly-lactide-glycolides), polyvinyl alcohols, gelatins, or
alginates. Such dosage formulations can be prepared, for example,
in the form of microsphere suspensions, gels (of hydrophilic or
hydrophobic constitution), or shaped-polymer matrix implants that
are well-known in the art for their function as "depot-type" drug
delivery systems that provide prolonged release of the biologically
active components. Such compositions can be prepared using
art-recognized formulation techniques and designed for any of a
wide variety of drug release profiles.
[0051] The administration of pharmaceutical compositions for use in
the present invention can be intermittent or at a gradual, or
continuous, constant or controlled rate to a patient in need of
treatment. In addition, the time of day and the number of times of
day that the pharmaceutical formulation is administered can vary
depending on the patient condition and environment. The level of
efficacy and optimal dosage and dosage form for any given peptidase
inhibitor for use within the scope of this invention is
patient-dependent and adjustable within reasonable ranges in the
judgment of the attending physician. The formulation is typically
administered over a period of time sufficient to treat or prevent
the patient disease state, e.g., to modify the behavioral or
cognitive performance of the patient undergoing treatment. The
peptidase inhibitor formulations may be continued to be
administered using the same or attenuated dosage protocol for
prophylaxis of the targeted disease state.
[0052] The above-described embodiments of the present invention
derive in part from the mechanism of action deduced from data
gathered in animal behavioral cognitive and skill models described
below. Other embodiments of the invention will be apparent from
analysis of the data obtained in the following non-limiting
experimental examples, which are but illustrative of the behavior
modification and cognitive performance and improvement attainable
by use of the method and formulations of the present invention.
Experimental Examples
[0053] Marketed in 1981-1982 moxalactam (Mox) was employed widely
in the world as a third-generation cephalosporin-like antibiotic.
Clinical efficacy and safety were evaluated in over 2200 patents
with bacterial infections (Jackson et al. 1986). Of the 260 patents
treated with Mox for gram-negative meningitis, 241 (93%) showed
satisfactory response to antibiotic therapy. Patents were treated
with 4 g of Mox every 8 hrs for 2-3 weeks. Peak plasma
concentrations occur within an one hr after IM injection with an
elimination half-life of 2.3 hrs. There is no accumulation with
multiple injections occurring at 8-12 hr intervals. Moxalactam can
penetrate the blood brain barrier. Cerebrospinal fluid (CSF) levels
of Mox range from 25-39 .mu.g/ml following a 2.0 g IV dose of drug.
The CSF concentration as a percentage of serum concentration is
estimated to be 20%. The D isomer has antibacterial activity and
has a greater unbound fraction to plasma protein that the L
isomer.
Behavioral Studies with Moxalactam
Methods
Animal Care
[0054] Male Syrian golden hamsters (Mesocricetus auratus) (140-150
g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis,
Ind.) were housed individually in Plexiglas cages (24 cm.times.24
cm.times.20 cm), maintained on a reverse light:dark cycle (14L:10D;
lights on at 19:00 hr) and provided food and water ad libitum.
Animals were acclimated to the reverse light:dark cycle for at
least two weeks before testing. All behavioral tests were conducted
during the dark phase of the circadian cycle. All animals were
acquired and cared for in accordance with the guidelines published
in the Guide for the Care and Use of Laboratory Animals (National
Institutes of Health Publications No. 85-23, Revised 1985).
Offensive Aggression
[0055] Agonistic behavior can be classified as either offensive or
defensive aggression (Blanchard and Blanchard, 1977; Adams, 19798;
Albert and Walsh, 1984). Offensive aggression is characterized by
the aggressor initiating an attack on an opponent, while defensive
aggression lacks active approach. Both types of aggression have
their own unique neurobehavioral systems. The stimuli that elicit
offensive and defense attack are different, as are the sequences of
behaviors that accompany each agonistic response. While much of the
empirical data supporting the notion of unique offensive and
defensive neural networks have been collected from animal models,
there are interesting and compelling similarities in human
aggression that suggest a similar neural organization (Blanchard,
1984). Offensive aggression is easily studied using male golden
hamsters tested in a resident/intruder paradigm, an established
model of offensive aggression (Ferris and Potegal 1988). Placing an
unfamiliar male hamster into the home cage of another male hamster
elicits a well-defined sequence of agonistic behaviors from the
resident that includes offensive aggression.
Behavioral Measures and Analysis
[0056] Hamsters are nocturnal and as such all behavioral tests were
performed during the first four hrs of the dark phase under dim red
illumination. The resident was scored for offensive aggression,
e.g., latency to bite the intruder, the total number of bites,
total contact time with the intruder and flank marking over a 10
min test period (Ferris and Potegal, 1988). Flank marking is a form
of olfactory communication in which a hamsters arches its back and
rubs pheromone producing flank glands against objects in the
environment (Johnson, 1986). Flank marking frequency is greatly
enhanced during aggressive encounters and is particularly robust in
dominant animals initiating and winning fights (Ferris et al.,
1987).
[0057] Parametric data, i.e., latencies and contact time, were
analyzed with a one-way ANOVA followed by Newman-Keuls post hoc
tests. Non parametric data, i.e., number of bites and flank marks,
were analyzed with Kruskal-Wallis tests followed by Mann-Whitney U
tests to determine differences between groups. Two sample
comparisons were analyzed with paired and unpaired t-Tests for
parametric data and Wilcoxon and Mann-Whitney Tests for paired and
unpaired non-parametric data, respectively.
Results
I. High Dose Moxalactam
[0058] In a pilot study, Mox (50 mg/kg in a volume of ca. 150
.mu.l) was given intraperitoneally (IP) to six male hamsters
prescreened for aggressive behavior toward smaller intruders.
Treatments with Mox and saline vehicle were counter balanced so
each animal received both treatments separated by at least 48 hr.
Animals were tested 90 min after treatment a period estimated to
reflect peak plasma levels of Mox (Jackson et al. 1986). Moxalactam
was dissolved in 0.9% NaCl and stored on ice. It was prepared fresh
for each study.
[0059] Resident animals treated with saline vehicle bite intruders
in less than one min (FIG. 1). Following Mox treatment the mean
latency to bite was increased to over six min (p<0.05). In
addition, the number of bites over the 10 min observation period
were significantly reduced (p<0.05). However, the contact time,
i.e., the time the resident spent smelling and exploring the
intruder was also significantly reduced (p<0.01). The decrease
in flank marking did not reach significance but there was a trend
(p<0.07).
SUMMARY
[0060] The general decrease in all behavioral measures associated
with offensive aggression raises the possibility that the 50 mg/kg
dose of Mox has non specific depressive effects on motor activity
and arousal. To examine this possibility, it was necessary to run
dose response studies to find the lowest dose of Mox that
effectively inhibits offensive aggression without altering other
behaviors.
II. Moxalactam Dose Response
[0061] To find the lowest dose of Mox that could significantly
reduce offensive aggression, a range of concentrations (vehicle,
0.5, 5.0, 50, 500, and 5000 .mu.g/kg) were tested in six animals
(FIGS. 2 & 3) The treatments were counter balanced with each
animal receiving each treatment separated by at least 48 hrs. The
latency to bite was significantly different between treatments (F
(5,30)=5.66; p<0.001). Moxalactam treatment with doses of 5.0
.mu.g and less had no effect on any behavioral measures of
offensive aggression. However, the dose of 50 .mu.g/kg
significantly delayed bite latency by over seven min (p<0.001)
as compared to vehicle control. Doses of 500 .mu.g and 5.0 mg also
significantly increased bite latency. As was expected, the same
doses that increased bite latency also decreased the number of
bites (H=24.12; p<0.001). Animals treated with 50 .mu.g Mox
showed a significant reduction in bites (p<0.05). Indeed, three
of six animals never bite at all in the 10 min observation period.
The contact time was significantly different between treatments (F
(5,30)=2.5; p<0.05). Doses of 500 .mu.g and 5 mg significantly
reduced contact time as compared to vehicle control (p<0.05 and
p<0.01, respectively). Flank marking was not significantly
different between groups (H=9.256; p<0.09).
SUMMARY
[0062] These data identify the dose of 50 .mu.g/kg of Mox as very
effective in inhibiting offensive aggression without significantly
reducing contact time and flank marking. Higher doses of Mox, while
effective in reducing measures of aggression also reduced contact
time. Hence, the 50 .mu.g dose would appear to be best for future
behavioral tests. Having identified the most effective dose of Mox
a more thorough study using a greater number of animals and a
greater spectrum of behavioral tests was necessary.
III. Behavioral Tests with 50 Mg Moxalactam
Offensive Aggression
[0063] Thirteen hamsters were tested for offensive aggression
following treatment with saline vehicle or 50 .mu.g/kg Mox (FIG.
4). Both treatments were given IP in a volume of ca. 150 .mu.l.
Animals were tested 90 min after injection. Each animal received
both treatments. The order of injections was counter balanced with
no less than 48 hrs between treatments. Moxalactam significantly
increased bite latency (p<0.001) and reduced the number of bites
(p<0.01). There was no significant change in contact time or
flank marking.
SUMMARY
[0064] This larger study of low dose Mox corroborates the
dose-response study confirming that Mox can effectively reduce
offensive aggression without altering social behavior as measured
by the time spent with the intruder.
Motor Activity in an Open Field
[0065] Six animals were tested for general motor activity in an
"open field" following treatment with saline vehicle or 50 .mu.g/kg
Mox (FIG. 5). The study was counter balanced with each animal
receiving each treatment. Ninety minues after injection a single
animal was placed into a large clean Plexiglas cage
(48.times.32.times.40 cm) devoid of any bedding. This open field
was delineated into equal quadrants by tape on the underside of the
cage. Animals were scored for motor activity by counting the number
of quadrants traversed in 1 min. There was no significant
difference between treatments on open field activity.
Olfactory Discrimination
[0066] Sixteen animals were treated with vehicle or 50 .mu.g/kg Mox
and tested for olfactory discrimination by measuring their latency
to find hidden sunflower seeds (FIG. 5). The injections were
counterbalanced with each animal receiving each treatment. Prior to
testing animals were fasted for 24 hrs. Ninety minutes after
injection animals were briefly taken from their home cage while six
sunflower seeds were buried under the bedding in one corner.
Animals were placed back into their home cage and scored for the
latency to find the seeds in a ten min observation period. The
latency to find the seed was significantly (p<0.001) reduced in
animals treated with Mox as compared to vehicle controls.
Surprisingly, all seeds were rapidly consumed in less than five min
following treatment with Mox but not saline. In fact, not one of
the sixteen animals consumed all of the seeds following saline as
compared to all animals treated with Mox.
Sexual Activity
[0067] Six animals were tested over a five min observation period
for sexual activity following treatment with saline vehicle or 50
.mu.g/kg Mox (FIG. 6). The study was counter balanced with each
animal receiving each treatment. Ninety min after injection,
animals were scored for latency to mount and number of
intromissions, i.e., bouts of copulation, toward a receptive female
placed into their home cage. Female golden hamsters were
ovariectomized under general anesthesia. Following recovery animals
were treated with a single SC injection of 50 mg estradiol benzoate
for three consecutive days to induce sexual receptivity. On the day
of testing the estrogen primed females were introduced into the
home cage of the experimental males. The first investigation by the
males routinely caused robust lordosis in the female. Lordosis, is
a stereotyped posture characterized by intense, sustained vertebral
dorsiflexion.
[0068] Following vehicle treatment, animals mounted and thrust a
receptive female in ca. 30 sec. The time to mount was significantly
increased (p<0.05) following treatment with Mox. While both
treatments showed high bout of copulation, animals treated with Mox
showed a trend toward a decreased intromission rate
(p<0.07).
SUMMARY
[0069] Moxalactam appears to have a very good serene profile.
Serenics are drugs used to treat impulsivity and violence (Olivier
and Mos, 1991). Serenics should suppress offensive aggression
without interfering with social, appetitive and cognitive
behaviors. Social interest in an intruder, i.e. contact time is not
altered by Mox. Flank marking and activity in an open field is also
unaltered with drug treatment evidence that general arousal and
motor activity is normal. Fasted animals treated with Mox are
better able to find hidden sunflower seeds evidence that drug
treatment does not interfere with olfaction or motivation to find
food; in fact, it may enhance it. Interestingly, Mox treatment
reduced the latency to mount a receptive female and lessened,
although not significantly, the bouts of copulation in a five min
observation period. It should be noted that Mox treated animals
were still very sexually active, except the behavior appeared less
intense. This antiaggressive effect of Mox combined with a
mollification of sexual activity might have therapeutic value in
treating violent sex offenders.
[0070] Development of eltoprazine, one of the first serenics, was
abandoned, in part, because it was found to increase fear and
anxiety in animals (Olivier et al 1994). To control for this
possibility, it was necessary to test Mox in a model used to screen
drugs for their affect on anxiety
IV. Testing Moxalactam for Anxiolytic Activity
Elevated Plus-Maze
[0071] The elevated plus-maze was developed for the detection of
anxiolytic and anxiogenic drug effects in the rat (Pellow et al.,
1985). The method has been validated behaviorally, physiologically,
and pharmacologically. The plus-maze consists of two open arms and
two enclosed arms. Rats will naturally make fewer entries into the
open arms than into the closed arms and will spend significantly
less time in open arms. Confinement to the open arms is associated
with significantly more anxiety-related behavior and higher stress
hormone levels than confinement to the closed arms. Clinically
effective anxiolytics e.g., chlordiazepoxide or diazepam,
significantly increase the percentage of time spent in the open
arms and the number of entries into the open arms. Conversely,
anxiogenic compounds like yohimbin or amphetamines reduce open arm
entries and time spent in the open arms.
Method
[0072] Male Wistar rats weighing 250-300 g were group housed in a
normal 12:12 light-dark cycle light on at 0800 hr and provide food
and water ad libitum. The plus-maze consisted of two open arms,
50.times.10 cm, and two enclosed arms 50.times.10.times.40 cm with
an open roof, arranged such that the two open arms were opposite to
each other. The maze was elevated to a height of 50 cm.
[0073] Eight animals were tested in the plus-maze 90 min following
IP injection with 50 .mu.g/kg Mox and saline vehicle. The order of
treatments was counter balanced with at least 48 hrs between
injections. At the start of the experiment the animal was place in
the center of the plus maze facing the closed arm. Over a five min
observation period, animals were scored for the latency to enter
the closed arm, time spent in the closed arm and the number of open
arm entries following the first occupation of the closed arm (FIG.
7). Treatment with Mox significantly increased the latency to enter
the closed arm (p<0.05) as compared to vehicle. The time spent
in the closed arm was significantly reduced (p<0.01), while the
number of open arm entries was significantly elevated
(p<0.05).
SUMMARY
[0074] These data show Mox given at a dose of 50 .mu.g/kg has
anxiolytic activity. This finding enhances the serenic profile of
Mox and delineates it from previous serenics like eltoprazine that
suppressed offensive aggression, in part, by increasing fear and
anxiety. These data also show that Mox may have therapeutic value
as an anxiolytic
[0075] However, the anxiolytic activity of Mox raises other
concerns about behavioral specificity. Many anxiolytics,
particularly the benzodiazepines are sedatives and can depress
general motor activity and may also acts as amnesics and interfere
with learning and memory. Since Mox was show to have no effect of
flank marking or activity in an open field it is unlikely to act as
a general sedative. However, it was necessary to test Mox for any
untoward effects on learning and memory.
V. Testing Moxalactam For Anxiolytic Activity Moxalactam V.
Chlordiazepoxide
Methods
[0076] Because Mox and CDP have different bioavailability profiles,
e.g. brain penetrance, their CNS activity could not be compared by
giving systemic injections of equimolar concentrations of each
drug. Instead it was necessary to give both drugs directly into the
cerebroventricular system to by pass the blood brain barrier.
Animals were anesthetized with sodium pentobarbital (50 mg/kg),
implanted with microinjection guide cannulae aimed at the lateral
ventricle and allowed to recover for two days before testing. To
groups of six animals each were tested with Mox or CDP. Each
animals received a injection of drug and 0.9% NaCl vehicle on two
separate days. The order of injections was counterbalanced and
separated by two days. Both Mox and CDP were prepared in 0.9% NaCl
at a concentration of 1 mM. All injections were given in a volume
of 2 ul over 10 secs in fully conscious, restrained animals. Sixty
min later animals were tested in the plus-maze for a 3 min
observation period and scored for behaviors as noted
previously.
Results
[0077] Mox treatment significantly (p<0.05) delayed the time it
took to enter the closed arm as compared to vehicle treatment (FIG.
17). Treatment with Mox caused animals to spend most of their time
in the light arms of the plus-maze. Time spent in the dark was
significantly (p<0.01) lower following Mox treatment as compared
to vehicle. Treatment of CDP at the 1 mM concentration had no
effect on either the latency to enter the closed arm or time spent
in the closed arm as compared to vehicle treatment.
Controlling for Non-Specific Depression of Motor Activity
[0078] When CDP is given systemically to rodents in doses of 5-15
mg/kg it is a sedative and depresses motor activity. However, this
depression of motor activity disappears following repeated
administration of CDP over several days. Only after the animals
become insensitive to the motor effects of CDP are they tested in
the plus maze for anxiolytic activity. To control for any
non-specific effects of Mox and CDP on motor activity following
their direct injection into the brain, animals were tested in the
open field 30 min prior to testing in the plus (FIG. 18). There was
no significant effect for either anxiolytic on general motor
activity.
SUMMARY
[0079] The finding that Mox is an anxiolytic enhances its serenic
profile and delineates it from previous serenics like eltoprazine
that suppressed offensive aggression, in part, by increasing fear
and anxiety. On an equimolar basis, Mox showed anxiolytic activity
given directly into the brain as compared to CDP which had none.
These data show that Mox may have therapeutic value as an
anxiolytic in addition to a serenic.
[0080] However, the anxiolytic activity of Mox raises other
concerns about behavioral specificity. Many anxiolytics,
particularly the benzodiazepines are sedatives and can depress
general motor activity and may also acts as amnesics and interfere
with learning and memory. Since Mox was show to have no effect of
flank marking or activity in an open field it is unlikely to act as
a general sedative. However, it was necessary to test Mox for any
untoward effects on learning and memory.
VI. Testing Moxalactam for Spatial Memory Radial Arm Maze
[0081] The radial arm maze is one of the most commonly used methods
for testing spatial learning and memory in rodents. Developed by
Olton and co-workers (1976), it provides the simultaneous choice of
several alternative paths for the test subject. Animals must learn
which locations provide food (place learning) using visuospatial
cues.
Methods
[0082] Experimental Trials: The experimental trials consist of
three phases (described below). The arms of the maze are numbered
clock-wise from one to seven with arm number one being the arm
furthest to the right side of the maze. All trials are ca. 12 min
long. When not being tested, all hamsters have unlimited access to
water. In addition to the sunflower seeds in the maze, hamsters are
given one Agway Prolab 3000 food pellet daily. Trials within all
the phases are conducted on successive days.
[0083] Phase One: Phase One consists of five 15 min trials. Prior
to the beginning of each of the five trials in Phase One, four
sunflower seeds are placed at the ends of arms one, two, and three.
Arms four, five, six, and seven remain empty.
[0084] Phase Two: Phase Two of the experimental trials are
identical to Phase One except that the seeds are placed in arms
two, four, and seven. Arms one, three, five and six remain empty.
Phase Two consists of four 15 min trials.
[0085] Phase Three Phase Three of the experimental trials consists
of three 15 min trials, with arm two, four, and seven baited with
sunflower seeds. Phase Three differ form Phase Two in that the maze
is rotated clockwise in the room 110.degree..
[0086] Coding of Behaviors: An arm entry was scored if all four
paws of a hamster crossed an arm threshold. A full arm entry into
an awl is scored if a hamster's snout touches the top of the block
at the end of an arm or if their snout passes the block. These
scores were made for baited and unbaited arms. In addition, the
number of seeds pouched by the hamsters was scored.
Results
[0087] Six male hamsters were tested in the radial arm maze
following treatment with 0.9% NaCl or 50 .mu.g/kg Mox (FIG. 8).
Each animal received each treatment and the order of treatments was
counter balanced. The most critical measure in the radial aim maze
is the number of seeds discovered after reversing the orientation
of the maze on the final day of testing. Moxalactam treatment
significantly increased seed finding (p<0.01) as compared to
vehicle treatment.
SUMMARY
[0088] These data support the notion that the anxiolytic profile of
moxalactam is not accompanied by any disruption in learning and
memory as is the case with benzodiazepine anxiolytics. On the
contrary, moxalactam enhances spatial memory would may act as a
psychotropic agent to improve cognitive performance. This finding
suggests that moxalactam may be an effective therapeutic agent for
the treatment of ADHD and conduct disorder in children and senility
in geriatric patients.
Spatial Navigation in Water Maze
[0089] The Morris water maze like the radial arm maze was developed
to test spatial memory (Morris, 1984). The pool is divided into
quadrants usually designated North, South, East and West. The water
in the pool is made opaque with milk powder. Hidden just beneath
the surface in one of the quadrants is a platform that serves as a
escape route for rodents placed into the pool. An animal is placed
some where in the pool from a variety of different start points and
is timed for latency to find the platform, percent time spent in
each quadrant, distance traveled and swimming speed. The animals
has no visual or spatial cues in the pool and must rely on
extra-maze cues, i.e., objects set up outside the pool that can be
seen by the swimming animal. Through a series of trials a rat
develops "place learning" or knowledge about the position of the
platform based upon the extra-maze cues. The platform can be moved
to a different quadrant each day combining spatial memory with
working memory. This paradigm involves extinction of the prior
memory and resolution of a new spatial problem.
Methods
[0090] The water maze consisted of a black plastic circular pool
ca. 150 cm in diameter and 54 cm in height filled to a level of 35
cm with water made opaque with powdered milk. The pool was divided
into four quadrants with a platform 10 cm in diameter submerged 2
cm below the surface in the northwest quadrant. The water was
maintained at a temperature of 25.degree. C. Around the pool were
several visual cues. Above the pool was a video camera for tracking
the movement of the experimental animal. The data collection was
completely automated using the software developed by HVS Image
(Hampton, UK). Before testing, rats were familiarized with the pool
and platform placed in the northwest quadrant. Each day for 4
consecutive days, animals were placed into pool at random sites and
given two min to find the platform. Animals were treated one hr
before testing with 50 .mu.g/kg Mox (n=11) or vehicle (n=10).
Following these familiarization trials, animals were tested for
spatial navigation. The first day of testing began with the
platform in the expected northwest quadrant. All behavior was
videotaped for a two min observation period. After testing the
animal were dried off and placed back into their home cage. On each
subsequent day the platform was moved to a new quadrant and the rat
started at different positions. The rat was always placed into the
pool facing the side wall. The start positions relative to the
platform were different for each of the four trials; however, the
platform was always in the same relative position in each quadrant.
Twenty cm in from the side of the pool and in the left corner from
the center facing out.
Results
[0091] A two-way ANOVA showed a significant main effect for
treatment (F.sub.(1,20)=6.48, p<0.05) and days of testing
(F.sub.(3,63)=5.76, p<0.01) (FIG. 19). There was also a
significant interaction between treatments and testing days
(F.sub.(3,63)=4.35, p<0.01). Newman-Keuls post hoc tests showed
a significant difference between treatments on day two (p<0.05),
day three (p<0.01) and day four (p<0.05) (FIG. 19). On each
of these days Mox treated animals showed significantly shorter
latencies to find the hidden platform than the vehicle treated
group. Indeed, vehicle treated animals showed a significant
increase in latency on days 2 (p<0.05) and 3 (p<0.01) as
compared to day 1.
[0092] The strategy for finding the platform was strikingly similar
for both treatments (FIG. 19, lower two graphs) as judged by the
percentage of time the animals spent in each quadrant. For any
quadrant on any day there was no significant difference between
treatments. There was a significant difference between days for
percentage of time spent in any particular quadrant (e.g., North,
F.sub.(3,63)=28.80, p<0.0001). Animals spent a significant
portion of their time in certain quadrants on certain days. For
example, on Day 1 both Mox and Vehicle animals spent most of their
time in the North quadrant as compared to the other quadrants
(p<0.01). This was to be expected since they had knowledge of
the location of the platform in this quadrant from the
familiarization procedure. Interestingly, Vehicle animals also
showed a significant (p<0.05) amount of time in the West
quadrant on Day 1 as compared to South and East. This was probably
because the platform was hidden in the northwest part of the North
quadrant. On Day 2, Mox and Vehicle animals spent a significant
amount of time in both the North and South quadrants as compared to
East and West. On Day 3 Mox animals show no particular bias for any
quadrant while Vehicle animals still show a significant interest in
the North quadrant as compared to South and West. By Day 4 both Mox
and Vehicle spent most of their time in the correct quadrant (West)
with the least amount of time in the East quadrant where the
platform was hidden the day before. This strategy on Day 4 shows
good spatial, working and procedural memory for both
treatments.
[0093] The distance covered to reach the platform across days was
not significantly different between Mox and Vehicle animals (FIG.
20). However, Mox animals showed significantly greater swim speed
than Vehicle animals (F.sub.(1,20)=22.94, p<0.0001)(FIG. 20).
For example, on Day 2 both groups traveled a similar distance to
the platform except Mox animals covered the distance at almost
twice the speed (p<0.01). While there was no main effect across
days (F.sub.(3,63)=2.27, p<0.09) there was an interaction
between swim speed and days (F.sub.(3,63)=2.75, p<0.05) for Mox
treatment as this group decreased their swim speed over time.
Cue Navigation in Water Maze Method
[0094] On the day following the last day (Day 4) of spatial
navigation, animals were tested for cue navigation. In these tests,
the platform was raised above water level. One hr before testing
animals were treated with Mox or saline vehicle. The same animals
that were treated with Mox during spatial navigation were treated
with Mox for cue navigation. Animals were run through a series of
two minute trials with 45 min between trials. At each trial, the
platform was moved to a different quadrant. The cue navigation
study was identical to the spatial navigation except the platform
was visible and the testing was done over five consecutive trials
done on a single day. Animals were scored for latency to find the
platform, percent time spent in each quadrant, path distance and
swim speed for all testing periods
Results
[0095] The latency to find the platform was different between Mox
and Vehicle treated animals (F.sub.(1,20)=24.68, p<0.0001) (FIG.
21). There was also a main effect for days (F.sub.(4,84)=6.53,
p<0.0001) but no interaction between treatment and days
(F.sub.(4,84)=0.99, p<0.4). On trials 1,3, and 4 Mox animals
showed significantly shorter latencies than Vehicle animals.
[0096] As in spatial navigation, the strategy for finding the
platform was very similar for both treatments (FIG. 21, lower two
graphs) as judged by the percentage of time the animals spent in
each quadrant. For any quadrant on any trial there was no
significant difference between treatments (e.g., South,
F.sub.(1,20)=1.61, p<0.21). There was a significant difference
between trials for percentage of time spent in any particular
quadrant (e.g., South, F.sub.(4,84)=16.70, p<0.0001). Animals
spent a significant portion of their time in certain quadrants on
certain trials. For example, on Trial 5 both Mox and Vehicle
animals spent a significant amount of time in the North quadrant
were the platform was hidden, and the West quadrant were the
platform had been on the previous trial.
[0097] Unlike spatial navigation, the distance traveled during cue
navigation was significantly different between Mox and Vehicle
animals (F.sub.(1,20)=44.11 p<0.0001) (FIG. 22). There was also
a significant main effect for trials (F.sub.(4,84)=7.90,
p<0.0001) and interaction between treatment and trails
(F.sub.(4,84)=2.67, p<0.05). On Trial 1 there was no difference
in path length between treatments. However, on Trials 3 and 4
Vehicle animals traveled significantly farther to find the platform
than Mox animals. The path length did not significantly change
across trials for Mox animals. Whereas, the mean path length on
Trial 3 for Vehicle animals was significantly greater than any
other trail for this treatment.
[0098] Unlike spatial navigation, there was no significant
difference in swim speed between the two treatments
(F.sub.(1,20)=0.67, p<0.42) (FIG. 22). However, there is a main
effect across trials (F.sub.(4,84)=17.18, p<0.0001) and an
interaction between treatment and trials (F.sub.(4,84)=4.10,
p<0.01). In both treatments there is a significant increase in
swim speed over each subsequent trail. For example, from Trial 1 to
Trial 4 Mox and Vehicle animals showed a significant increase in
swim speed (p<0.01).
SUMMARY
[0099] Moxalactam treated animals are more effective in finding the
hidden and visible platform in the water maze than vehicle treated
controls. However, the strategy for success in each navigation
paradigm was strikingly different. During spatial navigation,
animals must rely on extramaze cues and procedural memory to find
the moving platform. Mox and vehicle animals appeared to show the
same learning and memory as there was no difference in the
percentage of time spent in each quadrant for each day of testing.
There was no ostensible difference in the swim patterns (FIGS. 23
and 24). The distance traveled between treatments was not
significantly different. Mox animals found the platform sooner, in
part, because they swam faster. However, cue navigation presented a
different profile. Again Mox treated animals out performed vehicle
animals on latency to find the platform. Again the search strategy
as defined by the percentage of time spent in each quadrant was
strikingly similar. However, unlike spatial navigation, animals
treated with Mox showed a much shorter path length. Moreover, both
treatment groups swam at the same speed.
[0100] These data support the notion that the anxiolytic profile of
moxalactam is not accompanied by any disruption in learning and
memory as is the case with benzodiazepine anxiolytics. On the
contrary, moxalactam enhances spatial memory and may act as a
psychotropic agent to improve cognitive performance. This finding
suggests that moxalactam may be an effective therapeutic agent for
the treatment of ADHD and conduct disorder in children and senility
in geriatric patients.
VII. Social Behavior in Non-Human Primates Experimental
Procedure
[0101] Eight, two year old adolescent male rhesus macaques were
tested with Mox. Animals were raised with their mothers in a group
setting at a field station. At one year of age, they were
transferred to individual cages. Each day thereafter, they were
paired housed for two-three hrs. The adolescent partners were
always the same. This year long procedure resulted in adolescent
partners or "play-mates" having a well-defined history of social
interaction with recognizable dominant and subordinate status. The
display of social behaviors in this arrangement are very robust
because of the limited amount of time the monkeys spend
together.
[0102] During the experiment the monkeys were paired in the
"play-cage" where they were video taped for one hour. The study was
designed so that behavioral data were obtained for each monkey
under Mox and vehicle treatment. The treatment was an ABA type
schedule of administration: Day 1--one member of each pair received
0.9% NaCl vehicle, Day 2--drug, Day 3--vehicle. Only one member of
a pair was injected on a test day. The other member of a pair was
injected a week later according to the same ABA schedule.
Moxalactam was injected LM in a dose of 1 mg/kg. Animals were video
taped sixty minutes after injection for a one hr observation
period. Animals were scored for over forty different behaviors
(Winslow et al., 1988). Only twenty-eight are listed on TABLE I.
The unreported behaviors, e.g., self-bites, vocalizations,
clinging, mounts, escapes, self grooming were so infrequent that
they were omitted from the analysis. Paired t-test was run for each
behavioral measure.
Results
[0103] The duration of play fighting was significantly reduced
(p<0.05) by Mox treatment as compared to vehicle. This finding
was not affected by the social status of the animal, i.e. both
dominant and subordinate animals showed diminished play fighting
following treatment with Mox. Interestingly, several different
measures of agonistic behavior, e.g., composite aggression scores,
clustered together at near significant levels. It should be noted
that these are juvenile rhesus monkeys, and as such their
expression of social aggression is primarily confined to play
fighting. The aggression does not have the same emotional valence
as adults. Nonetheless play fighting is thought to be the juvenile
antecedent to adult aggression. Allogrooming for adolescent and
adult monkeys is the primary measure of affiliative behavior. While
Mox significantly reduced the duration of play fighting it had no
effect on allogrooming.
SUMMARY
[0104] Moxalactam given in a dose of 1 mg/kg to adolescent rhesus
monkeys significantly reduces play fighting a measure of agonistic
behavior. However, allogrooming the key measure of affiliative
behavior is unaltered. Hence the finding that Mox can reduce
agonistic behavior in rodents translates to non-human primates.
VIII. Testing D and L Isomers of Moxalactam Rationale
[0105] The 3D structure of drugs can naturally occur as mirror
images or isomers. These isomers are classified as D or L based on
their rotation of light. Only one of the isomers usually has
biological activity. Since the preparation of Mox used in these
studies is a mixture of the two isomers it was necessary to
isolated and test for the active isomer.
Methods
[0106] Moxalactam sodium salt (FW 564.4) was obtained as a mixed
isomer from Sigma Chemical (St Louis Mo.). D, L-Mox were isolated
with HPLC using the method outlined by Ziemniak et al., 1982.
D,L-Mox was taken up in water and fractioned on a C18 column with a
running buffer of 1% MeCN, pH 6.5. Column effluent was monitored at
275 nm with a UV detector. Both isomers came out as single peaks. D
Mox had a retention time of 6.7 min while L-Mox came out at 8.2
min. The individual isomers of Mox provided to be relatively
unstable and would rapidly re-isomerize during lyophilization
making it difficult to have a reasonably pure (>98%) sample.
Hence it was necessary to go directly from the HPLC to the animal.
D isomer (ca. 200 .mu.g/ml HPLC buffer) was diluted to 50 .mu.g/ml
saline and keep on ice until IP injection (50 .mu.g/kg). L isomer
(ca. 150 .mu.g/ml HPLC buffer) was also diluted to 50 .mu.g/ml
saline and treated similarly.
Results
[0107] Two groups of eight animals each were tested for offensive
aggression following treatment with 50 .mu.g/kg D or L Mox (FIG.
9). Animals were tested 90 min after injection. D Mox significantly
increased bite latency (p<0.01) and reduced the number of bites
(p<0.05). There was no significant difference in contact time or
flank marking between the two isomers.
SUMMARY
[0108] These data identify D moxalactam as the active isomer
affecting offensive aggressive behavior.
IX. Testing Beta-Lactam Related Antibiotics for Antiaggressive
Effects Rationale
[0109] Moxalactam is chemically and pharmacologically similar to
cephalosporin and penicillin antibiotics. Indeed, moxalactam is
classified as a cephalosporin. The basic structures of all
cephalosporins and penicillin are show below. Each has a
beta-lactam ring (A), in turn, cephalosporin has a six-sided
dihydrothiazine ring (B) and penicillin a five-sided thiazolidine
ring (B). These basic structures that form the chemical nucleus for
these antibiotics occur naturally in fungus. Moxalactam is not
found in nature and is characterized by an oxygen substitution for
the sulfur (S) atom in cephalosporin.
##STR00003##
[0110] Cephalosporins and penicillin are bacteriocidal. Their
antibacterial activity is due to an inhibition of peptidoglycan
synthesis in the bacterial cell walls. Although the exact mechanism
of action is not fully understood, these antibiotics bind to
several proteolytic enzymes, e.g., carboxypeptidases and
endopeptidases, that are involved in synthesizing the peptidoglycan
latticework that strengthens the bacterial cell wall. The
interaction between these antibiotics and the proteolytic enzymes
is reversible. It is thought that these beta-lactam antibiotics act
as substrate analogs for acyl-D-alanyl-D-alanine, the endogenous
substrate for these enzymes. When these bacterial enzymes are bound
up with antibiotic they cannot perform their function and the
bacteria lyse as they replicate.
[0111] Similar carboxypeptidases and endopeptidases are associated
with cell membranes of neurons and glia in the mammalian brain. One
of their many functions is to rapidly degrade neuropeptides acting
as neurotransmitters. Unlike the classical neurotransmitters, e.g.
dopamine and serotonin, that rely on reuptake mechanisms to stop
signal activation, neuropeptides are inactivated by their rapid
degradation in the extracellular space. These beta-lactam related
antibiotics are believed to have psychotropic activity by
interfering with the metabolism (NAALADase activity) on the
numerous neuropeptides altering the neuropeptide milieu of the
brain.
Method
[0112] Six animals were tested with equimolar concentrations (90
.mu.M) of Moxalactam (Mox), Ampicillin (Amp) Carbenicillin (Carb)
Cefoxitin (Cef), Amoxicillin (Amox) or saline vehicle. The
concentrations were adjusted to equal the 50 .mu.g/kg dose used for
MOX in previous studies. All solution were prepared in 0.9% NaCl
and given IP. The order of injections was counter balanced. Animals
were tested for offensive aggression 90 min after injection (FIG.
10). There was a significant difference between treatments on bite
latency (F (5,30)=2.83; p<0.05). Both Mox and Amp significantly
delayed the latency to bite (p<0.001 and p<0.05,
respectively) as compared to vehicle control. There was also a
significant difference between treatments on number of bites
(H=10.6; p<0.05). Both Mox and Amp drugs significantly reduced
the number of bites (p<0.05). There were no significant
treatment effect on contact time or flank marking (FIG. 11).
SUMMARY
[0113] These data indicate that the antiaggressive effect of the
beta-lactam antibiotic Mox may be extended to include the
beta-lactam ampicillin. Of all of the antibiotics tested, Mox has
the greatest penetrability into the CNS. Patents given 2.0 g of Mox
IV show cerebrospinal fluid levels of drug around 30 .mu.g/ml. The
ratio of CSF to serum levels of Mox is ca. 15-20%. It is estimated
that the serum concentration of Mox in 140 g hamster given an IP
injection of 14 .mu.g of drug is 0.1 ng/ml. This would be reflected
by a CSF concentration of 15 ng/ml or brain levels of Mox
approximating 30 nM. These levels would certainly be in range to
interact effectively with neuropeptide receptors most of which have
binding affinities in the nanomolar range. Interaction with the
classical neurotransmitters would be less likely because these
receptors have Kd's in the micro and millimolar range.
[0114] Neonates with meningitis (conditions favoring CNS
penetrability of beta-lactam antibiotics) show a ratio of CSF to
serum level of Amp of ca. 10%. Cefoxitin, on the other hand has
poor CNS penetrability even when the meninges are inflamed. Perhaps
many of the beta-lactam antibiotics would be effective in
suppressing aggressive behavior and they are simply limited by
their pharmacokinetics and CNS penetrability. To test this notion
it was necessary to repeat the beta-lactam antibiotic study using a
higher dose of each drug.
X. High Dose Beta-Lactams
[0115] Six animals were tested with equimolar concentrations (ca. 5
mg/kg; 9 mM) of Ampicillin (Amp) Carbenicillin (Carb) and Cefoxitin
(Cef) or saline vehicle. The concentrations were adjusted to equal
the 5 mg/kg dose used in the dose response study for Mox. All
solution were prepared in 0.9% NaCl and given IP. The order of
injections was counter balanced. Animals were tested for offensive
aggression 90 min after injection (FIG. 12). There was a
significant difference between treatments on bite latency (F
(4,25)=5.49; p<0.01). Both Amp and Carb significantly delayed
the latency to bite (p<0.001) as compared to vehicle control.
There was also a significant difference between treatments on
number of bites (H=11.7; p<0.05). Both Amp and Carb
significantly reduced the number of bites (p<0.05 and p<0.01,
respectively). There were no significant treatment effect on
contact time or flank marking (FIG. 13).
[0116] Amoxicillin was not included in this high dose beta-lactam
antibiotic study; instead, it was run in a separate study using a
dose of 1 mg/kg (ca. 2 mM). Eight animals were tested for offensive
aggression 90 min after IP injection following treatment with Amox
or saline vehicle (FIG. 14). Each animal was given each treatment
with no less than 48 hrs between injections. The treatments were
counterbalanced. Aggressive behavior was not significantly altered
in animals treated with 1 mg/kg Amox.
SUMMARY
[0117] These data indicate that ampicillin and carbenicillin given
in high enough doses can suppress offensive aggression without
altering contact time or flank marking. These data raise the
possibility that the psychotropic effect of moxalactam is shared by
other beta-lactams and that the biological mechanisms of action may
be similar. Bioavailability and CNS penetrability, in part, may be
the major component contributing to differences in biological
efficacy. Indeed, more recent testing demonstrated that clavulanic
acid, a .beta.-lactam compound having no clinically significant
antibiotic activity, but a clinically important .beta.-lactamase
inhibition activity, exhibits a wide variety of psychotropic
effects, including antianxiety, antiaggression and cognition
enhancement, at i.p. doses less than 1 .mu.g/kg. Its high oral
absorption and good blood brain barrier transport properties make
it and related .beta.-lactamase inhibitors preferred candidates for
use in the methods and the pharmaceutical formulations in
accordance with this invention.
[0118] The mechanism (s) of action for the psychotropic effects of
these beta-lactams is now believed to be their interaction with
neurogenic NAALADase. This is feasible since cephalosporins are
reported to have bactericidal activity in concentrations as low as
10 nM. Note, the estimated concentration of Mox in the brain
following the 50 .mu.g/kg treatment is ca. 30 nM.
[0119] Another possible explanation for the psychotropic activity
of beta-lactam antibiotics is the possible blockade of known
neurotransmitter receptors or re-uptake proteins. To test this
second possibility it was necessary to screen Mox for receptor
interaction in a wide range of radio ligand binding assays.
XI. Screening Moxalactam in Receptor and Transport Binding Assays
Testing Mox For Vasopressin V.sub.1a And Serotonin 5ht.sub.1a
Receptor Interaction
[0120] Vasopressin and serotonin are both critical
neurotransmitters in the control of offensive aggression in male
hamsters (Ferris et al., 1998). These two neurotransmitters also
are implicated in the control of human aggression (Coccaro et al.,
1998). Vasopressin facilitates aggressive behavior while serotonin
inhibits aggression, in part, by inhibiting the activity of the
vasopressin system. Blockade of vasopressin V.sub.1A receptors and
stimulation of serotonin 5HT.sub.1A receptors in the anterior
hypothalamus blocks offensive aggression (Ferris et al., 1999).
Since Mox significantly suppresses offensive aggression it was
hypothesized it did so by interacting with either one or both of
these receptors. To test this notion Mox was tested in a membrane
binding assay for competition for the V.sub.1A receptor (Ferris et
al., 1994) and in a receptor autoradiography assay for competition
for the 5HT.sub.1A receptors (Ferris et al., 1999). Moxalactam in a
concentration of 1 .mu.M did not significantly displace I.sup.125
HO-LVA (vasopressin ligand) binding in a hamster liver membrane
preparation. Similarly, Mox was ineffective in reducing specific
binding of I.sup.125 DPAT (serotonin ligand) to tissue sections of
the hamster brain.
SUMMARY
[0121] These data show that moxalactam has no direct interaction
with vasopressin V.sub.1A and serotonin 5HT.sub.1A receptors in the
hamster. This would suggest that moxalactam is affecting behavior
by altering the activity of other neurochemical pathways.
Testing for Amino Acid, Adrenergic, Serotonergic, and Dopaminergic
Receptors And Their Transporters
[0122] Moxalactam was screened in thirty-six different binding
assays by NOVASCREEN, a contract research organization based in
Hanover, Md. Moxalactam was tested at 100 nM and nm in duplicate
samples for each of the assays listed on the following page. These
assays were chosen because their respective receptor or transporter
may play a role in the pathophysiology of mental illness.
Moxalactam had no significant effect in any of these binding
assays.
TABLE-US-00004 Amino Acid Targets Benzodiazepine, peripheral GABA
Agonist Site Benzodiazepine, central GABA Glutamate AMPA Site
Kainate Site NMDA, Agonist Site NMDA, Glycine
[strychnine-insensitive] site Glycine [strychnine-sensitive] site
Biogenic Amine-Adrenergic Targets Adrenergic .alpha..sub.1A
.alpha..sub.1B .alpha..sub.2A (human HT-29 cells) .alpha..sub.2B
.alpha..sub.2C (human recombinant) .beta..sub.1 .beta..sub.2
Biogenic Amine-Serotonergic Targets Serotonin 5HT.sub.1A (human
recombinant) 5HT.sub.1B 5HT.sub.1D 5HT.sub.2A (formerly 5HT.sub.2)
5HT.sub.2C 5HT.sub.3 5HT.sub.4 5HT.sub.6 (rat recombinant)
5HT.sub.7 (rat recombinant) Biogenic Amine-Dopaminergic Targets
Dopamine D.sub.1 D.sub.2 (human recombinant) D.sub.3 (rat
recombinant) Clozapine Uptake/Transporter Targets Adrenosine
Adrenergic, Norepinephrine Dopamine GABA Glutamate Muscarinic,
Choline Serotonin Hormone Targets Corticotropin Releasing
Factor
Testing for Corticotropin Releasing Hormone Receptor
[0123] Corticotropin releasing hormone (CRH or CRF as shown on the
following page) is a critical neurohormone in the regulation of
stress. Since Mox suppresses impulsivity, aggression, and anxiety
while enhancing learning and memory it may be acting to reduce
stress. For this reason, Mox was tested by NOVASCREEN in a CRF
binding assay. Moxalactam at a concentration of 100 nM had no
effect in this assay.
SUMMARY
[0124] These data show that moxalactam does not interact directly
with many of the receptors and transporters implicated in the
pathophysiology of aggression and mental illness. This leaves three
possible mechanisms of action: 1) interaction with known receptors
that were not screened, e.g., histamine, acetylcholine, and other
neuropeptides, 2) interaction with unknown or "orphan receptor," or
3) interaction with peptidolytic enzymes (e.g., NAALADase) in the
CNS that alter the chemical milieu of the brain.
XII. Examining Mechanism of Action
Testing Peptidoglycan-Precursor Peptide For Effects On Offensive
Aggression Rationale
[0125] The beta-lactam antibiotics have a stereochemistry that
resembles acyl-D-alanyl-D-alanine, the natural substrate for the
bacterial proteolytic enzymes. Presumably, this structural
characteristics enables beta-lactam antibiotics to behave as
competitive substrate blocking enzyme activity. To test this
hypothesis an analog of acyl-D-alanyl-D-alanine,
peptidoglycan-precursor peptide (Nieto and Perkins 1971; Zeiger and
Maurer, 1973) was tested for antiaggressive effects in the hamster
resident/intruder paradigm.
Method
[0126] Peptidoglycan-precursor peptide,
Ala-D-.gamma.-Glu-Lys-D-Ala-D-Ala, (PPP) was obtained from Sigma
Chemical and reconstituted in DMSO and diluted in 0.9% NaCl to a
final concentration of ca. 2 mM. Animals were anesthetized with
sodium pentobarbital (50 mg/kg), implanted with microinjection
guide cannulae aimed at the lateral ventricle and allowed to
recover for two days before testing. On the day of testing, animals
(n=6) were injected with vehicle (2% DMSO in 0.9% NaCl) or PPP in a
dose of ca. 1 mg/kg in a volume of 1 .mu.l Sixty minutes after
injection, animals were retested for offensive aggression toward a
smaller intruder placed into their home cage. Two days later
animals were tested again and the order of treatments reversed.
Results
[0127] Peptidoglycan-precursor peptide significantly increased the
latency to bite (p<0.05) and reduced the number of bits
(p<0.05) during a 10 min. Observation period (FIG. 15). There
was no significant difference in contact time or flank marking
between treatments (FIG. 15).
Testing Peptidoglycan-Precursor Peptide for Effects of Olfactory
Discrimination
[0128] Six animals received an intracerebroventricular injection of
vehicle or 1 mg/kg PPP and tested for olfactory discrimination by
measuring their latency to fid hidden sunflower seeds (FIG. 16).
The injections were counterbalanced with each animal receiving each
treatment. Prior to testing animals were fasted for 24 hrs. Sixty
min. After injection animals were briefly taken from their home
cage while six sunflower seeds were buried under the bedding in one
corner. Animals were placed back into their home cage and scored
for the latency to find the seeds in a five min. Observation
period. The latency to find the seed was significantly (p<0.05)
reduced in animals treated with PPP as compared to vehicle.
SUMMARY
[0129] The direct injection of peptidoglycan-precursor peptide into
the brain of hamsters has the same behavioral results as the
peripheral injection of Mox. Both drugs and both routes of
administration significantly reduce aggressive behavior without
altering social interest of motor activity, i.e., contact time and
flank marking. In addition, the enhancement of olfactory
discrimination that appears to be the simplest and most robust
behavioral assay for screening beta-lactam antibiotics is similarly
affected by the precursor peptide. These findings are evidence that
beta-lactam antibiotics affect behavior by: 1) acting directly on
the brain, and 2) resembling the acyl-D-alanyl-D-alanine peptide
moiety.
[0130] While clavulanic acid contains a beta-lactam ring and is
structurally similar to penicillins and cephalosporins, it has weak
antibacterial activity with no therapeutic value as an antibiotic.
However, when given in combination with some beta-lactam
antibiotics like ticarcillin (Timentin.RTM.) clavulanic acid can
extend the spectrum and enhance the activity of the antibiotic
(AHFS, 1991). This synergistic activity is possible because
clavulanic acid acts as an irreversible competitive inhibitor of
bacterial beta-lactamases that naturally degrade and inactive
beta-lactam antibiotics (Brown et al., 1976; Reading and Cole
1977).
##STR00004##
[0131] Clavulanic acid is commercially available in the United
States but only in fixed combination with other drugs. Commonly
prescribed Timentin.RTM. is normally given intravenously in doses
ranging from 200-300 mg/kg/day (based on ticarcillin content) which
corresponds to a dose of clavulanic acid of approximately 7-10
mg/kg/day (AHFS, 1991). There are no reported adverse reactions or
contraindications for clavulanic acid given in this dose range
(Koyu et al., 1986; Yamabe et al., 1987). The data presented below
report clavulanic acid can alter CNS activity and behavior at doses
ranging from 10 ng to 10 .mu.g/kg, or 1000 to 1,00,000 times less
than used in antibacterial indications.
[0132] Clavulanic acid by itself is orally active and stable. The
bioavailability is approximately 64 to 75% (Davies et al., 1985;
Bolton et al., 1986) with an elimination half-life of just under
two hours. Peak plasma concentrations occur between 45 min to three
hours after ingestion (Bolton et al., 1986) with a plasma half-life
of over 2 hrs (Nakagawa et al., 1994). The volume of distribution
is around 15 liters suggesting clavulanic acid is primarily
confined to extracellular fluid (Davies et al., 1985). The
CSF/plasma ratio is around 0.25, evidence that clavulanic acid
readily passes the blood-brain barrier (Nakagawa et al., 1994).
Behavioral Studies with Clavulanic Acid
I. Clavulanic Acid Dose-Response in the Seed Finding Model of
Anxiety
[0133] Clavulanic acid (CLAV) is structurally similar to the
beta-lactam antibiotics. A most robust and simple bioassay for
screening beta-lactams for CNS activity is the golden hamster seed
finding model of anxiety. Briefly, hamsters are deprived of food
overnight. The following day they are exposed to the additional
stress of being taken from their home cage and placed in a novel
environment for a few minutes. This manipulation stimulates the
release of the stress hormone cortisol (FIG. 37). During their
absence from the home cage, sunflower seeds are hidden under the
bedding in one of the corners. When returned to the home cage,
hamsters routinely scramble along the walls for 1-2 min before
settling down, locating and eating the seeds. However, animals
treated with the benzodiazepine anxiolytic chlordiazepoxide find
seeds in less than 10 sec. This reduction in seed finding time from
minutes to seconds also occurs following treatment with moxalactam
and other beta-lactam antibiotics.
Experimental Protocol
[0134] Male, Syrian golden hamsters (Mesocricetus auratus) (120-130
g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis,
Ind.) were housed individually in Plexiglas cages (24 cm.times.24
cm.times.20 cm), maintained on a reverse light:dark cycle (14L:10D;
lights on at 19:00 hr) and provided food and water ad libitum. A
range of concentrations of CLAV (saline vehicle, 0.1, 1.0, 10, 100
1,000 ng/kg) wire tested in six groups of hamsters (4-8/group)(FIG.
25). All tests were conducted during the dark phase of the
circadian cycle under dim red illumination. Prior to testing all
animals were fasted for 20-24 hrs. Ninety min after intraperitoneal
(IP) injection of drug, animals were taken from their home cage and
placed into a holding cage for 2 min. During their absence, six
sunflower seeds were buried under the bedding in one corner of
their home cage. Animals were placed back into their home cage
randomly facing any one of the empty corners and timed for their
latency to find the seeds in a five min observation period. Latency
times were analyzed with a one-way ANOVA followed by Scheffe's post
hoc tests. Assumption of equal variances was tested (Hartley's
F-max=2.1 p>0.05)
Results
[0135] The latency to find the sunflower seeds was significantly
different between doses (F.sub.(5,30)=10.0; p<0.0001). CLAV in
doses of 10 ng and above significantly (p<0.01) reduced latency
times to less than 8.0 sec as compared to saline vehicle with a
mean latency of 104 sec. The dose of 1 ng/kg was not significantly
different from vehicle control.
SUMMARY
[0136] The data show CLAV given in a dose of 10 ng/kg body weight
has maximal efficacy the seed finding test. The adult male hamsters
used in these studies weighed around 125 g. Hence, these animals
were given about 1.25 ng of CLAV. CLAV has a volume of distribution
approximating the extracellular fluid volume. The extracellular
water content of lean body mass is approximately 22%. The
concentration of 1.25 ng of CLAV in 27.5 ml of water is 0.045 ng/ml
or about 200 pM (formula weight of the potassium salt of CLAV is
ca. 240). Since the CSF/plasma ratio is 0.25 the estimated
concentration in the brain would be around 50 pM.
[0137] The seed finding model of anxiety appears to have empirical
validity (McKinney 1989) i.e., drugs like benzodiazepines that are
used to treat clinical anxiety are effective in the animal model.
However, a wider spectrum of anxiolytics and non-effective drugs
must be screened to assess the incidence of false negatives and
false positive before adopting seed finding as a model of anxiety.
Hence, it was necessary to validate the potential anxiolytic
activity of CLAV in the traditional elevated plus-maze.
II. Testing Clavulanic Acid in the Elevated Plus-Maze
[0138] The elevated plus-maze was developed for screening
anxiolytic and anxiogenic drug effects in the rat (Pellow et al.,
1985). The method has been validated behaviorally, physiologically,
and pharmacologically. The plus-maze consists of two open arms and
two enclosed arms. Rats will naturally make fewer entries into the
open aims than into the closed arms and will spend significantly
less time in open arms. Confinement to the open arms is associated
with significantly more anxiety-related behavior and higher stress
hormone levels than confinement to the closed arms. Clinically
effective anxiolytics, e.g., chlordiazepoxide or diazepam,
significantly increase the percentage of time spent in the open
arms and the number of entries into the open arms. Conversely,
anxiogenic compounds like yohimbin or amphetamines reduce open arm
entries and time spent in the open arms.
[0139] Male Wistar rats weighing 250-300 g were group housed in a
normal 12:12 light-dark cycle with light on at 0800 hr and provided
food and water ad libitum. The plus-maze consisted of two open
arms, 50 cm long, 10 cm wide, with walls 40 cm high made of clear
Plexiglas. The two closed arms had the same dimensions but included
a roof. The Plexiglas for the closed arms was painted black. Each
pair of arms was arranged opposite to each other to form the
plus-maze. The maze was elevated to a height of 50 cm. Eighteen
animals were tested in the plus-maze 90 min following the IP
injection of 1.0 .mu.g/kg CLAV, 50 or vehicle control in a volume
of ca. 0.3 ml. The order of treatments was counter balanced with at
least 48 hrs between injections. At the start of the experiment,
the animal was placed at the end of one of the open arms. Over a
five min observation period, animals were scored for the latency to
enter the closed arm, time spent in the closed arm and the number
of open arm entries following the first occupation of the closed
arm. The study produced tables of repeated measures. The data
between treatments were compared with a two-way, repeated measures
ANOVA followed by Bonferroni post hoc tests. There was a
significant difference between treatments for latency to enter the
dark (F.sub.(1,18)=8.53; p<0.01). When treated with CLAV
(p<0.05) animals stayed in the starting open light position
longer than when treated with vehicle (FIG. 26). The time spent in
the open arm was highly significant between treatments
(F.sub.(1,18)=144; p<0.0001) (FIG. 26). The time spent in the
open arm was significantly increased for CLAV (p<0.01) as
compared to vehicle. Finally, the open arm entries were
significantly different between treatments (F (1, 18)=44.0
p<0.0001) with CLAV (p<0.01) treatment showing increased
movement into the lighted open arms as compared to vehicle (FIG.
26).
[0140] These data show CLAV given at a dose of 1 .mu.g/kg has
anxiolytic activity in the plus-maze. These data are encouraging;
however, many anxiolytics such as the benzodiazepines depress motor
activity. Since animals treated with CLAV took a longer time to
move from the lighted open arm to the dark, protected, closed arm
it could be argued that this beta-lactam did not reduce anxiety,
instead it sedated the animal and retarded movement. To control for
this possibility it was necessary to screen CLAV for general motor
activity in an open field paradigm.
III. Motor Activity in an Open Field
Experimental Protocol
[0141] Immediately after each of the plus-maze tests reported above
in Section II, animals were tested for general motor activity in an
"open field." Animals were placed into a large clean Plexiglas cage
(48.times.32.times.40 cm) devoid of bedding. This open field was
delineated into equal quadrants by tape on the underside of the
cage. Animals were scored for motor activity by counting the number
of quadrants traversed in 1 min. There were no significant
differences between CLAV and vehicle treatment on open field
activity (FIG. 27).
SUMMARY
[0142] There is no evidence in the open field test that CLAV
depress motor activity. This finding is corroborated in another
behavioral study, flank marking reported in Section VII. Flank
marking is a complex stereotyped motor behavior used by hamsters to
disseminate pheromones for olfactory communication (FIG. 39). Flank
marking is unaffected by treatments with CLAV. It would appear that
this beta-lactam has an advantage over the more conventional
benzodiazepine anxiolytics since it does not depress motor
activity. However, is the anxiolytic activity of CLAV comparable to
the clinically prescribed benzodiazepines?
IV. Clavulanic Acid Vs Chlordiazepoxide in the Plus-Maze
Experimental Protocol
[0143] Chlordiazepoxide)(Librium.quadrature. is a commonly
prescribed anxiolytic that has been thoroughly characterized in
preclinical studies. The effective anxiolytic dose in the plus-maze
is 10-25 mg/kg (Lister 1987; File and Aranko 1988; Shumsky and
Lucki 1994). In this range of doses, chlordiazepoxide (CDP) is a
sedative and depresses motor activity complicating the
interpretation of any behavioral assay that requires locomotion
(McElroy et al., 1985). However, it was discovered animals develop
a tolerance to the motor depression with repeated daily
administration of CDP for several days (Shumsky and Lucki 1994).
Hence in these studies, rats (n=6) were given a single IP injection
of CDP (10 mg/kg) each day for seven days prior to the start of the
experiment. While CLAV has no effect on motor activity it was
necessary to treat an equal number of rats with daily injections of
CLAV (100 ng/kg) to insure a balanced experimental design. In
addition there was a third group of rats (n=6) receiving daily
injections of saline vehicle. The study reported in Section II
tested CLAV at 1 .mu.g/kg in the plus-maze. The data from the seed
finding assay of anxiety shown in Section I suggests CLAV should be
effective between doses of 10 ng to 1 .mu.g/kg. For this reason
CLAV was tested at 100 ng/kg in these studies.
Results
[0144] There was a significant difference between treatments
(F.sub.(2,15)=21.45, p<0.001) for the latency to enter the dark.
The latency to enter the dark closed arms was significantly greater
for animals treated with CLAV and CDP (p<0.01) as compared to
vehicle control (FIG. 28A). There was also a significant difference
between treatments (F.sub.(2,15)=17.14, p<0.001) for the time
spent in the light. The time spent exposed to light in the open
arms was also significantly greater for the CLAV and CDP
(p<0.01) treated animals as compared to vehicle (FIG. 28A).
There was no significant difference between treatments for open arm
entries (FIG. 28B).
SUMMARY
[0145] These data show that CLAV and CDP have similar anxiolytic
activity in the elevated plus-maze. Yet, CLAV has greater potency
being effective at a dose 100,000 times less than CDP. Furthermore,
CLAV does not have the sedative, motor depressant activity of the
conventional benzodiazepine anxiolytics. The anxiolytic effects of
CLAV are immediate and do not require the development of tolerance
to realize behavioral efficacy. However, a point of caution,
benzodiazepines have another undesirable side effect for which
there is no development of tolerance-amnesia (Shumsky and Lucki
1994). For example, diazepam)(Valium.degree. selectively impairs
short-term memory and attention while sparing long-term memory
(Liebowitz et al., 1987; Kumar et al., 1987). Hence, it was
necessary to test CLAV for any untoward effects on learning and
memory.
V. Clavulanic Acid and Spatial Memory in the Water Maze
[0146] The Morris water maze was developed to test spatial memory
(Morris, 1984). The pool is divided into quadrants usually
designated North, South, East and West. The water in the pool is
made opaque with milk powder. Hidden just beneath the surface in
one of the quadrants is a platform that serves as a escape route
for rodents placed into the pool. An animal is placed some where in
the pool from a variety of different start points and is timed for
latency to find the platform, percent time spent in each quadrant,
distance traveled and swimming speed. The animals have no visual or
spatial cues in the pool and must rely on extra-maze cues, i.e.,
objects set up outside the pool that can be seen by the swimming
animal. Through a series of trials a rat develops "place learning"
or knowledge about the position of the platform based upon the
extra-maze cues. The platform can be moved to a different quadrant
each day combining spatial memory with working memory. This
paradigm involves extinction of the prior memory and resolution of
a new spatial problem.
Methods
[0147] The water maze consisted of a black plastic circular pool
ca. 150 cm in diameter and 54 cm in height filled to a level of 35
cm with water made opaque with powdered milk. The pool was divided
into four quadrants with a platform 10 cm in diameter submerged 2
cm below the surface in the northwest quadrant. The water was
maintained at a temperature of 25.degree. C. Around the pool were
several visual cues. Above the pool was a video camera for tracking
the movement of the experimental animal. The data collection was
completely automated using the software developed by HVS Image
(Hampton, UK). Before testing, rats were familiarized with the pool
and platform placed in the northwest quadrant. Each day for 4
consecutive days, animals were placed into pool at random sites and
given two min to find the platform. Animals were treated one hr
before testing with 1.0 .mu.g/kg CLAV (n=9) or vehicle (n=9).
Following these familiarization trials, animals were tested for
spatial navigation. The first day of testing began with the
platform in the expected northwest quadrant. All behavior was
videotaped for a two min observation period. After testing the
animal were dried off and placed back into their home cage. On each
subsequent day the platform was moved to a new quadrant and the rat
started at different positions. The rat was always placed into the
pool facing the sidewall. The start positions relative to the
platform were different for each of the four trials; however, the
platform was always in the same relative position in each quadrant.
It was positioned 20 cm in from the side of the pool and in the
left corner from the center facing out. The latency to find the
hidden platform, path length, swim rate, and quadrant times between
CLAV and vehicle treated animals were compared with a two-way,
repeated measures ANOVA followed by Bonferroni post hoc tests.
Results
[0148] There was no main effect for drug treatment
(F.sub.(1,16)=4.17, p<0.057), days of testing
(F.sub.(3,48)=0.51, p>0.5) or interaction between factors
(F.sub.(3,48)=1.92 p>0.1) (FIG. 29) for latency to find the
platform. However, animals treated with CLAV showed shorter
latencies to find the platform on Days 1 and 4 with a trend towards
significance.
[0149] The strategy for finding the platform was similar for both
treatments (FIGS. 30A & B) as judged by the percentage of time
the animals spent in each quadrant. For any quadrant on any day
there was no significant difference between treatments. There was a
significant difference between days for percentage of time spent in
any particular quadrant (e.g., CLAV, North Quadrant,
F.sub.(3,32)=38.81, p<0.0001). Animals spent a significant
portion of their time in certain quadrants on certain days. For
example, on Day 1 both CLAV and vehicle animals spent most of their
time in the North quadrant as compared to the other quadrants
(p<0.01). This was to be expected since they had knowledge of
the location of the platform in this quadrant from the
familiarization procedure.
[0150] While the strategy for finding the platform as measured by
percentage of time spent in each quadrant was similar between CLAV
and vehicle there was a small but obvious difference. Animals
treated with CLAV spent more time in the correct quadrant than
animals treated with vehicle. This difference is particularly true
on Day 2 when the CLAV animals spent over 50% (p<0.01) of their
time in the correct (South) quadrant. The vehicle animals spent
less than 40% of their time in the correct quadrant, a time not
significantly different from the other quadrants. By Day 4 both
CLAV and vehicle spent most of their time in the correct quadrant
(West). This strategy on Day 4 shows good spatial, working and
procedural memory for both treatments.
[0151] There was a significant main effect for treatment
(F.sub.(1,16)=8.40, p>0.01) on the path length to find the
platform. On Day 1 CLAV treated animals (p<0.05) traveled a much
shorter distance during the search for the platform than vehicle
animals (FIG. 31). There was no significant difference between CLAV
and vehicle on swim rate (FIG. 32).
2. Cue Navigation
Method
[0152] On the day following the last day (Day 4) of spatial
navigation, animals were tested for cue navigation. In these tests,
the platform was raised above water level. One hr before testing
animals were treated with CLAV or saline vehicle. The same animals
treated with CLAV during spatial navigation were treated with CLAV
for cue navigation. Animals were run through a series of two min
trials with 45 min between trials. At each trial, the platform was
moved to a different quadrant. The cue navigation study was
identical to the spatial navigation except the platform was visible
and the testing was done over five consecutive trials done on a
single day. Animals were scored for latency to find the platform,
percent time spent in each quadrant, path distance and swim speed
for all testing periods
Results
[0153] There was no main effect for treatments (F.sub.(1,16)=0.553
p>0.1), trials (F.sub.(4,64)=0.9745, p>0.1) or interaction
between factors (F.sub.(4,64)=0.7433, p>0.5) for latency to find
the platform during cue navigation (FIG. 33).
[0154] As in spatial navigation, the strategy for finding the
platform was very similar for both treatments (FIGS. 34A & B)
as judged by the percentage of time the animals spent in each
quadrant. For any quadrant on any trial there was no significant
difference between treatments (e.g., Trial 1, North,
F.sub.(1,16)=0.099, p>0.5). There was a significant difference
for percentage of time spent in any particular quadrant for either
treatment for most of the trials, most notably for CLAV.
[0155] The distance traveled to find the platform was not
significantly different between CLAV and vehicle animals
(F.sub.(1,16)=0.23 p>0.5) (FIG. 35). While there was no
significant main effect for treatment on swim rate
(F.sub.(1,16)=0.926, p>0.1), there was a significant trails
effect (F.sub.(4,64)=7.87, p<0.001) and interaction between
factors (F.sub.(4,64)=2.56, p<0.05). Both treatments, but
particularly CLAV showed reduced swim rates by Trial 4 (p<0.01)
and Trial 5 (p<0.05). This probably reflects the fact that they
knew where to look for the platform as shown in FIGS. 34A &
B.
SUMMARY
[0156] Clavulanic acid treated animals do not show any loss in
learning and memory when tested for spatial and cue navigation in
the Morris water maze. Indeed, on distance traveled to the hidden
platforms and percentage of time spent in the correct quadrant for
both spatial and cue navigation, CLAV treated animals showed better
performance than vehicle. These data show that the anxiolytic
profile of CLAV is not accompanied by any disruption in learning
and memory as is the case with benzodiazepine anxiolytics.
VI. Clavulanic Acid and the Stress Response
[0157] The ability of CLAV to reduce anxiety in stressful
situations, i.e. the food deprivation and novel environment in the
seed finding assay, and exposure to light and a novel environment
in the elevated plus-maze, without altering motor activity or
cognitive function is a significant finding. The potential of CLAV
as an anxiolytic and therapeutic in the treatment of numerous
affective disorders could be broadened if we had a clearer
understanding of its mechanism of action. For example, could CLAV
be altering anxiety by suppressing the natural stress response? The
commonly prescribed benzodiazepine anxiolytics block both the
normal circadian release and stress-mediated release of the hormone
cortisol (Gram and Christensen, 1986; Petraglia et al., 1986;
Hommer et al., 1986).
[0158] The simple procedure of placing an adult male hamster into a
novel environment for 5 min causes a significant, predictable
increase in blood levels of cortisol (Weinberg and Wong 1986). This
novelty test was used to assess the effects of CLAV on
stress-induced release of cortisol. Two groups of male hamsters
were treated IP with either CLAV (10 .mu.g/kg, n=6), or saline
vehicle (n=4). A third group (n=4) received no treatment or
isolation stress and served as a control for basal levels of
cortisol. Sixty min after treatment animals were taken from their
home cage and placed into a novel cage for 5 min. Afterwards
animals were sacrificed by decapitation and trunk blood collected
for radioimmunoassay of cortisol. All animals were tested under
reverse light:dark conditions four hrs into the dark cycle. Data
were compared with a one-way ANOVA followed by Fisher PLSD post hoc
tests.
[0159] There was a significant difference in the stress release of
cortisol between treatments (F.sub.(2,11)=10.03 p<0.01). Vehicle
(p<0.05) and CLAV (p<0.01) showed more than twice the blood
level of cortisol as compared to the untreated, non-stressed
control (FIG. 37).
[0160] The data show that the beta-lactam anxiolytic CLAV has no
ostensible effect on the release of cortisol in response to the
mild stress of exposure to a novel environment. This detail,
combined with the absence of motor depression and cognitive
impairment makes CLAV unique amongst the anxiolytics and suggests a
highly specific, novel mechanism of action. At first glance one
might think it would be advantageous to suppress the stress
response. Indeed, hypercortisolism has been implicated in the
pathophysiology of depression (Sacher et al., 1973). Chronic
psychosocial stress leading to dysfunctional, hyperactive adrenal
glands can be life threatening. However, a responsive
hypothalamic-pituitary-adrenal axis is critical for normal
physiology and behavior. Stressors that would normally help animals
adapt to the environment can be fatal without the appropriate
release of cortisol.
VII. Territorial or Offensive Aggression
[0161] Continuing to study the CNS activity of CLAV in more complex
behavioral models may help to clarify its mechanism(s) of action.
For example, antagonistic, social interactions between animals
require risk assessment, communicative and agonistic behaviors to
settle disputes over territory, mates, food, etc. The
neurotransmitters serotonin and vasopressin are fundamental in the
CNS organization and expression of these behaviors in animals and
humans (Ferris et al., 1997; Coccaro et al., 1998; Ferris 2000). To
this end, CLAV was tested for effects on territorial or offensive
aggression, i.e. defense of the home burrow against intruders.
[0162] Agonistic behavior can be classified as either offensive or
defensive aggression (Blanchard and Blanchard, 1977; Adams, 19798;
Albert and Walsh, 1984). Offensive aggression is characterized by
an aggressor initiating an attack on an opponent; while, defensive
aggression lacks active approach. Both types of aggression have
their own unique neurobehavioral systems. The stimuli that elicit
offensive and defense attack are different, as are the sequences of
behaviors that accompany each agonistic response. While much of the
empirical data supporting the notion of unique offensive and
defensive neural networks have been collected from animal models,
there are interesting and compelling similarities in human
aggression that suggest a similar neural organization (Blanchard,
1984). Offensive aggression is easily studied using male golden
hamsters tested in a resident/intruder paradigm, an established
model of offensive aggression (Ferris and Potegal 1988) in the
context of defending the home burrow. Placing an unfamiliar male
hamster into the home cage of another male hamster elicits a
well-defined sequence of agonistic behaviors from the resident that
includes offensive aggression. Hamsters are nocturnal and as such
all behavioral tests were performed during the first four hrs of
the dark phase under dim red illumination. The resident was scored
for offensive aggression, e.g., latency to bite the intruder, the
total number of bites, total contact time with the intruder and
flank marking over a 10 min test period (Ferris and Potegal, 1988).
Flank marking is a form of olfactory communication in which a
hamsters arches its back and rubs pheromone producing flank glands
against objects in the environment (Johnston, 1986). Flank marking
frequency is greatly enhanced during aggressive encounters and is
particularly robust in dominant animals initiating and winning
fights (Ferris et al., 1987).
[0163] Five male golden hamsters (130-140 g) were given IP
injections of CLAV (200 .mu.g/kg) and saline vehicle in a volume of
ca. 0.2 ml. In pilot studies, it was discovered CLAV given IP at
1.0 .mu.g/kg had no effect on aggressive behavior. Hence, it was
necessary to test CLAV at a higher concentration but in a dose
range that was still acceptable for pharmaceutical studies on
aggressive behavior. Vehicle and CLAV treatments were counter
balanced and randomized so all five animals received each treatment
separated by at least 48 hrs. Animals were tested 90 min after
treatment over a 10 min observation period. Latencies and contact
time were analyzed with a two-way ANOVA. Non-parametric data, i.e.,
number of bites and flank marks were analyzed by Wilcoxon
matched-pairs signed-ranks test.
[0164] While there was no significantmain effect for drug treatment
(F.sub.(1,3)=7.40, P<0.07) for latency to bite the intruder
there was a trend toward significance (FIG. 38). There was no
significant main effect for drug treatment (F.sub.(1,3)=2.85,
p>0.1) on contact time with the intruder (FIG. 38). There was a
significant difference between drug treatments (T=3.0, p<0.05,
N=8) and the number of bites on the intruder. CLAV treatment
reduced the median number of bites to six as compared to thirteen
for vehicle treated animals (FIG. 39). There was no significant
effect of drug treatment (T=4.0, p>0.1, N=5) on the resident's
flank marking behavior (FIG. 39).
[0165] Clavulanic acid hasmodestantiaggressive or serenic-like
properties. Serenics are drugs used to treat impulsivity and
violence (Olivier and Mos, 1991). Serenics should suppress
offensive aggression without interfering with social, appetitive
and cognitive behaviors. Social interest in an intruder, i.e.
contact time was not altered by CLAV. Development of eltoprazine,
one of the first serenics, was abandoned, in part, because it was
found to increase fear and anxiety in animals (Olivier et al.,
1994). The potent anxiolytic activity of CLAV excludes this
possibility.
VIII. Interactions with Glutamyl Carboxypeptidase
[0166] CLAV has a very high binding affinity for the
beta-lactamases. It is hypothesized that the presence of mammalian
homologies to these bacterial enzymes and that these homologous
proteins are involved in the regulation of neurotransmitter levels
in the CNS. E Coli TEM beta lactamase has been cloned sequenced and
crystilized to determine the active site motifs. The four putative
binding sites on beta lactamase that could accommodate CLAV are
designated active site I, II, III, and IV. These active sites,
sequence location, and amino acid (AA) sequences are as
follows:
TABLE-US-00005 Site I: 35 AA's downstream from N-terminus: STTK
(SEQ ID NO: 1) Site II: 57 AA's downstream from STTK (SEQ ID NO: 1)
motif: SGC, SGN, or SAN Site III: 111 AA's downstream from SGC
motif: KTG Site IV: 41 AA's downstream from SGC motif: ENKD (SEQ ID
NO: 2)
[0167] Screening for amino acid sequence homologies between these
beta-lactamase binding sites and mammalian enzymes, Revaax
scientists identified an enzyme system in the brain that CLAV would
potentially bind in a similar manner to beta-lactamase. The enzyme
glutamyl carboxypeptidase (N-acetyl, alpha linked, acidic
dipeptidase) or NAALADase (Pangalos et al, 1999) is responsible for
regulating the glutamatergic neurotransmission pathways whose
effects would be expressed in such behavioral outcomes as
aggression, memory/cognition, and anxiety. As a result of the
almost perfect overlap of the putative active sites of
beta-lactamase and the conserved sequences in human and rat
NAALADase, it was hypothesized that CLAV affects behavior by
inhibiting NAALADase activity. The overlap sequence similarity
between beta-lactamase and NAALADase as shown below:
TABLE-US-00006 Site I: Beta-lactamase: 35 AA's downstream from
N-terminus: STTK (SEQ ID NO: 1) NAALADase: 38 AA's downstream from
N-terminus: STQK (SEQ ID NO:3) Site II: Beta-lactamase: 57 AA's
downstream from STTK (SEQ ID NO: 1) motif: SGC, SGN, or SAN
NAALADase: 59 AA's downstream from STQK (SEQ ID NO: 3) motif: SFG
Site III: Beta-lactamase: 111 AA's downstream from SGC motif: KTG
NAALADase: 110 AA's downstream from SFG motif: KLG Site IV:
Beta-lactamase: 41 AA's downstream from SGC motif: ENKD (SEQ ID NO:
2) NAALADase: 41 AA's downstream from SFG motif: ERGV (SEQ ID NO:
4)
[0168] Clavulanic acid inhibits gram negative beta-lactamase
enzymes in the range of 15-34 nM. CLAV is effective at a dose of 10
ng/kg in the seed finding model of anxiety (pg 3). If NAALADase
were the human homologue to beta-lactamase, then CLAV would be
predicted to be a high affinity substrate.
IX. Seed Finding Following Blockade of Naaladase Activity
[0169] It was hypothesized that CLAV functioned as an anxiolytic in
the seed finding assay by blocking NAALADase activity in the brain.
If this notion were true then it would be predicted that drugs
known to block NAALADase should also enhance seed finding. To this
end, animals were treated with N-acetyl-beta-aspartyl-glutamic acid
(beta-NAAG), a competitive inhibitor of NAALADase (Serval et al.,
1992) and tested in the seed finding model of anxiety. The study
was similar to that outlined in Section I with one notable
exception. Since beta-NAAG does not readily cross the blood-brain
barrier, it had to be injected directly into the lateral ventricle
where it could be carried by cerebrospinal fluid throughout the
brain via the ventricular system. Beta-NAAG (FW 304) was given in a
dose of 3 ng in a volume of 1 .mu.l saline ICV. The average adult
hamster brain weights ca. 1.2 g of which 22% is extracellular
fluid. The estimated beta-NAAG concentration was 11 ng/ml or 36
nM.
[0170] Two groups of six animals each were fasted overnight as
previously described and tested the following day. One group was
treated with beta-NAAG and the other saline vehicle and one hour
later timed for latency to find the hidden sunflower seeds. A
Student t-test for unpaired data was used for statistical
comparisons.
[0171] The difference in latency to find the seeds was
significantly (p<0.001) different between treatments (FIG. 40).
Indeed, the none of the six animals microinjected with saline
vehicle found the seeds in the five minute observation period.
However, three days later when these same animals were
microinjected with beta-NAAG (3 ng/.mu.l) and tested for seed
finding they showed a mean latency of 21.8.+-.9.7 sec. The data
show that beta-NAAG a specific NAALADase inhibitor, can
dramatically reduce the latency to find hidden sunflower seeds, a
biological activity shared by CLAV. Since beta-NAAG was active in
the seed finding model of anxiety, then the hypothesis that
beta-NAAG and CLAV share a common mechanism of action is not
rejected. From these data the hypothesis can be expanded to predict
that beta-NAAG and CLAV show similar effects on a range of
biological and behavioral measures. To this end, animals were
tested for offensive aggression in the resident intruder paradigm
as described in Section X. As reported earlier, when given in high
concentrations, CLAV has only a modest effect on offensive
aggression. While CLAV can enhance seed finding at a dose of 10
ngkg it has only a modest effect on offensive aggression even with
doses as high as 200 .mu.g/kg. If beta-NAAG and CLAY share a common
mechanism then beta-NAAG should have little or no effect on
aggression.
X. Effect of NAALADase Blockade on Offensive Aggression
[0172] The animals tested in this study were those used in Section
IX. After the seed finding assay, beta-NAAG (n=6) and saline
vehicle (n=6) treated animals remained in their home cage and were
presented with a smaller, male intruder. The resident was scored
for latency to bite, bites, contact time and flank marking over a
10 min observation period. Latency to bite and contact time between
treatments were compared with Student t-tests. Non-parametric
measures of bites and flank marks for beta-NAAG vs vehicle were
compared with Mann-Whitney.
[0173] There were no significant differences between beta-NAAG and
vehicle-treated animals for any measures of offensive aggression
(FIGS. 41 & 42).
[0174] Blocking NAALADase activity with beta-NAAG does not alter
offensive aggression as tested in the resident intruder paradigm.
This finding is not inconsistent with the notion that CLAV and
beta-NAAG share a common mechanism-blockade of NAALADase activity.
Sequence CWU 1
1
614PRTHomo sapiens 1Ser Thr Thr Lys124PRTHomo sapiens 2Glu Asn Lys
Asp134PRTHomo sapiens 3Ser Thr Gln Lys144PRTHomo sapiens 4Glu Arg
Gly Val154PRTHomo sapiens 5Asn Ser Arg Lys164PRTHomo sapiens 6Glu
Arg Gly Ile1
* * * * *