ES2370260T3 - NEUROTHERAPEUTIC COMPOSITION THAT INCLUDES A BETA-LACTAMA COMPOUND. - Google Patents

Abstract

A β-lactam compound selected from: i) clavulanic acid or a pharmaceutically acceptable salt thereof; or ii) moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof, for use in the treatment of a patient in need of relief from a selected disorder of anxiety, depression, dementia, Alzheimer's disease or Parkinson's disease

Description

Neurotherapeutic composition comprising a beta-lactam compound

Field of the Invention

The present invention relates to a novel mechanism of neuropsychiatric intervention. More particularly, the present invention relates to pharmaceutical formulations for the treatment of a variety of neurological disease states, including cognitive and behavioral disorders.

Background and summary of the invention

The pharmaceutical industry has directed extensive research and development efforts towards the discovery and commercialization of drugs for the treatment of neurological disorders. Such disorders usually derive from chemical imbalances in the brain. Excessive production or defective production of relevant neurochemical species and / or receptor dysfunction have been identified with many disease states recognized by neurologists, psychiatrists, psychologists and other doctors who are experts in the diagnosis and treatment of mental illness. Most of the effort for the discovery of new neurologically active drugs has been based on the study of the interaction of agonist / antagonist drugs with one or more of the numerous receptors in the brain and / or their respective receptor ligands.

The present invention provides a novel approach to drug intervention in the treatment of a wide variety of neurological disease states and other disease states or related clinical etiology states as defined in claim 1. It is based in part on the discovery. that compounds containing β-lactam known for their activity as inhibitors of bacterial peptidases or proteases, particularly transpeptidases and / or carboxypeptidases, are also potent inhibitors of certain mammalian neuropeptidases, including acidic N-acetylated-linked peptidases (NAALADases) ), several of which have been identified / characterized in the literature [Pangalos et al., J. Biol. Chem., 1999, 274, No. 13, 84708783]. The present invention is also based in part on the discovery that neurogenic NAALADases can be chosen as targets with NAALADase inhibitors to effect a significant modification of behavior and enhanced cognitive performance. Preliminary studies have confirmed that one or more neurogenic proteases, which are now believed to be NAALADases and related peptidases and transferases that can recognize and transform certain neuropeptides (e.g., N-acetyl-L-aspartyl-L-glutamate), play a role. significant, if not dominant, at the neurochemical level of brain function and concomitantly have a substantial impact on the patient's behavior and cognitive performance. It has previously been reported that certain glutamate analogs that act as NAALADase inhibitors can be used to treat prostate disease and glutamate abnormalities associated with certain nerve tissue trauma. It has now been determined that NAALADase inhibitors, which particularly include certain bacterial peptidase and β-lactamase inhibitors containing β-lactam capable of transporting the blood brain barrier, can act in the brain at very low concentrations as potent neuroactive active ingredients for reduce the symptoms of a wide variety of neurological disorders characterized by behavioral aberration or sensory / cognitive dysfunction. Significantly, it is believed that such bacterial enzyme inhibitors are effective inhibitors of NAALADase and neurogenic peptidases related to concentrations below those concentrations known to be required for the clinically effective inhibition of bacterial enzymes. Therefore, it is expected that such compounds can also be used effectively to treat prostate disease and disease states associated with trauma to nerve tissue previously described as being sensitive to treatment with other NAALADase inhibitors.

The present invention relates to a β-lactam compound selected from:

i) clavulanic acid or a pharmaceutically acceptable salt thereof; or

ii) moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof,

for use in the treatment of a patient in need of relief from a selected disorder of anxiety, depression, dementia, Alzheimer's disease or Parkinson's disease and a pharmaceutical formulation for treating anxiety, depression, dementia, Alzheimer's disease or Parkinson's disease in a human patient in need of relief thereof, in which the formulation comprises a β-lactam compound selected from:

i) clavulanic acid or a pharmaceutically acceptable salt thereof; or

ii) moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof.

Accordingly, an embodiment of the present disclosure relates to a method for the treatment of cognitive and behavioral disorders in warm-blooded vertebrates by administering compounds known for their activity as protease inhibitors or bacterial peptidases, compounds that, when present at Effective concentrations in the brain, it has now been determined that they can inhibit or otherwise modulate the activity of one or more neurogenic NAALADases and related neurogenic enzymes.

In a related embodiment, a procedure for the treatment of cognitive and behavioral disorders in a patient in need of such treatment is disclosed. The method comprises the step to inhibit neurogenic peptidases that include NAALADase and related neurogenic enzymes. In one embodiment, such inhibition of neuropeptidases is effected by administering an effective amount of a β-lactam compound recognized for its ability to bind to and inhibit a bacterial enzyme, for example, a β-lactamase or a bacterial protease that participates in the synthesis of the bacterial cell wall. Such bacterial proteases are known in the art as "penicillin binding proteins." In another embodiment of the present disclosure, the process is performed by administration of NAALADase inhibitors recognized in the art that particularly include certain deaminoglutamate analogs and N-substituted glutamate derivatives. It has been found that the effective inhibition of such neuropeptidase activity in warm-blooded vertebrates produces a marked enhancement in cognitive performance and behavioral control.

By way of example, cognitive and behavioral disorders susceptible to treatment according to the present invention include anxiety and depression. Animal data suggest that the procedure and formulation have potential as an anti-aggressive agent to control impulsivity and violence in autism, Tourette syndrome, mental retardation, psychosis, obsession, senile dementia and individuals with personality disorders and a history of inappropriate aggression. Clinical 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 improve disorientation.

In another embodiment a method is provided for treating a patient suffering from a condition, or prone to developing a condition, characterized at least in part by abnormal extracellular concentration of glutamate in the brain or other nerve tissue. The method comprises the step of administering to the patient effective amounts of a compound that can inhibit the activity of a penicillin binding protein of bacterial origin. The composition is administered in amounts effective to prevent or alleviate the symptoms of such a condition. Therefore, for example, high concentrations of glutamate located in the brain have been reported in stroke victims and victims of other brain trauma. More recently, high concentrations of glutamate in the brain and peripheral nerve tissue that are associated with multiple sclerosis have been reported.

In yet another embodiment a method is provided for treating prostate disease selected from prostate cancer and benign prostatic hyperplasia in a human patient. The method comprises the step of administering to the patient a composition comprising a compound that can inhibit the activity of a penicillin binding protein of bacterial origin. The compound is administered in an amount effective to delay the evolution of the disease or to reduce the symptoms of the disease.

A group of compounds for use according to the present invention are β-lactam compounds, that is, compounds that have a β-lactam ring system that includes clavulanic acid and moxalactam. Non-β-lactam NAALADase inhibitors have been reported in the patent and non-patent literature. See, for example, US Pat. No. 5,795,877; 5,804,602; 5,968,915; 5,902,817; 5,962,521, 5,863,536 and 6,017,903, for their teaching of such NAALADase inhibitors and the generally use of such NAALADase inhibitors for the treatment of certain disease states sensitive to NAALADase inhibition therapy. Antibiotic compounds for use in the present invention can be administered in combination with one or more other enzyme inhibitors, for example, effective amounts of a β-lactamase inhibitor (wherein the active compound is a β-lactam compound) or another NAALADase inhibitor or a P-glycoprotein efflux inhibitor to boost levels of the active compound in the brain. The process and formulation embodiments of the invention are used in both human and veterinary health applications, for example, in canine, feline and equine species.

The description reveals that a warm-blooded vertebrate, most commonly a human patient, affected by a state of neurological disease characterized by cognitive or behavioral abnormalities is treated with a 1-oxa-1-detia-cephalosporin, more preferably a 7- methoxy-1-oxa-1-detia-cephalosporin, optionally as an active ester derivative in a formulation administered orally (including oral or sublingual administration) or parenterally. In one embodiment, the peptidases inhibitor is moxalactam, [7-β- [2-carboxy-2- (4-hydroxyphenyl) acetamido] -7α-methoxy-3 - [[(1-methyl-1H-tetrazol-5- il) thio] methyl] -1-oxa-1-detia-3-cefem-4-carboxylic], described and claimed with related compounds, which includes its active ester derivatives orally absorbed; in US Pat. No. 4,323,567 it has been found that moxalactam exhibits sensitive neuroactivity at significant doses when administered parenterally at least at approximately 50 µg / kg body weight.

In another embodiment of the present invention a pharmaceutical formulation is provided for the treatment with the consequent reduction of symptoms of behavioral or cognitive disorders in patients in need of such treatment as defined in claim 13. The formulation comprises a compound characterized by its affinity for bacteria derived from penicillin binding proteins. In one embodiment, the compound can bind to and inhibit the function of a bacterial protease known to exhibit its proteolytic activity on a peptidoglycan substrate comprising the C-acyl-D-alanyl-D-alanine C-peptide sequence. In another embodiment, the compound can bind to β-lactamase, another bacterial protein that can bind penicillin and inhibit the function of such an enzyme. The amount of the inhibitor used in the formulation is determined to be effective in inhibiting the activity of endogenous NAALADase. In one embodiment, the amount is effective to inhibit NAALADase in the brain at a level sufficient to modulate cognitive and behavioral characteristics. In this last embodiment, the level of activity presented by the NAALADase inhibitor in the present process not only depends on its affinity for penicillin-binding proteins and for NAALADase, it is also particularly dependent on the ability of the inhibitor compound to pass through the blood brain barrier to achieve effective levels in the brain to modify the patient's behavioral and / or cognitive performance.

In one embodiment of the invention, the pharmaceutical formulation comprises a compound containing β-lactam selected from the group consisting of clavulanic acid and moxalactam. In cases where the β-lactam compound is, for example, a commercially available antibiotic, the amount of the β-lactam compound in said formulation will be less than that required to produce, after administration by the commercially mode of administration. Detailed, clinically effective antibiotic blood levels of the compound. Reduced dosage levels of such antibiotics can still be effective, assuming reasonable transport properties to the blood brain barrier, to produce sufficient levels in the brain and CSF of the compound to inhibit neurogenic protease activity in the brain and modify cognitive characteristics. and of behavior. In addition, such formulations may optionally include effective amounts of one or more of a β-lactamase inhibitor and an efflux pump inhibitor of P-glycoprotein or other compound that can inhibit the activity of NAALADase and related neurogenic enzymes. While the formulations of the present invention can be specifically prepared for any mode of administration recognized in the art that can reach the inhibitory concentrations of threshold proteases in the brain, they are usually formulated for parenteral or oral administration, optionally in the form of prolonged release. or "slow release drug" formulations well known in the art.

Brief description of the drawings

Figs. 1-42 are graphical representations of data gathered in the performance of the tests of moxalactam, other antibiotics of β-lactam, clavulanic acid and other neuroactive compounds in various animal models accepted in the field for the detection of activity against offensive aggression. (Fig. 1-4, 9-14, 24, 29, 31 and 32), general motor activity, olfactory discrimination (Fig. 5), sexual activity (Fig. 6), anxiolytic activity (Fig. 7, 25, 26 , 28, 37 and 40) and spatial memory (Fig. 8 and 29-36). Fig. 15 and 16 are graphic illustrations of the effect of peptidoglycan precursor protein administered intracerebrally on offensive aggression and olfactory discrimination in hamsters.

Detailed description of the invention

The present invention and the various embodiments described and claimed herein derive, in part, from the findings that compounds that can bind to and inhibit the enzymatic activity of penicillin-binding proteins of bacterial origin are also potent inhibitors of the activity of N-acetylated α-linked acid peptidases (NAALADase) and possibly other related enzymes in the brain, and that when administered to provide effective threshold inhibition concentrations of NAALADase in the brain, NAALADase inhibitors have clinically significant neuroactivity evidenced in part by the modification of behavior and enhanced cognition and function.

In one embodiment, the NAALADase inhibitors effective for use according to the present invention are characterized by their ability to inhibit a bacterial protease that exhibits selective proteolytic activity in a peptide protein or substrate comprising acyl-D-alanyl-D-alanine. Alternatively, NAALADase inhibitors effective for use in the treatment of behavioral and cognitive disorders according to an embodiment of the present invention can be characterized by their selective affinity (for associative and / or covalent binding) to penicillin-binding proteins; Based on animal trials to date, such bacterial protease inhibitors appear to work at subclinical antibiotic levels in the brain to inhibit the activity of neuropeptidases that can function in neurochemical mediation of behavior and cognitive performance. Effective inhibition of neuropeptidases activity with concomitant mediation of behavior and cognitive performance has also been effected by the administration of a β-lactamase inhibitor, clavulanic acid, a compound containing β-lactam that has non-clinically significant antibiotic activity. . It is assumed that the inhibition of such neuropeptidases activity (for example, NAALADase) allows the modulation of the concentration and / or function of one or more neurotransmitters or neuromodulators with concomitant improvement in neurological function evidenced by the enhancement of cognitive performance and attenuation of aberrant behavioral phenotypes. In an example of the present invention, the administered moxalactam i.p. at 50 micrograms / kg it inhibits hamster aggression, enhances spatial learning in rats and acts as an anxiolytic in rats. Clavulanic acid has shown similar activity when i.p. at less than 1 microgram / kg.

Historically, those informed in the field of beta-lactam antibiotics understand that the mode of action as antibacterial agents is to inhibit the synthesis of the cell wall by acting as a substrate for penicillin-binding protein (PBP); The term PBP has been extended to include binding to all beta-lactams that include cephalosporins. More recently, researchers have been able to clone and sequence these PBPs, in addition to crystallizing enzymes and determining active site motifs (see P. Palomeque et al., J. Biochem., 279, 223-230, 1991). Based on these data, the four putative binding sites for PBP have been designated active site I, II, III and IV. The active sites, sequence location and amino acid sequence (AA) are as follows:

Active site I:

35 AA in the 3 'direction from the N: STTK end

Active site II:

57 AA at address 3 'from the STTK reason: SGC, SGN or SAN

Active site III:

111 AA in the 3 'direction from the SGC reason: KTG

Active site IV:

41 AA at address 3 'from the SGC reason: ENKD

According to the identification of an enzyme system in the brain that would inhibit moxalactam in a manner similar to PBP, it was discovered that a glutamyl-carboxypeptidase enzyme known as α-N-acetylated acid dipeptidase (NAALADase) (see MN Pangalos and col., J. Bio. Chem., 264, 8470-8483, 1999) has an almost perfect overlap of putative active PBP sites. This enzyme system 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 overlapping of putative active PBP sites and sequences conserved in human and rat NAALADase, it is believed that moxalactam and other β-lactam compounds mediate behavioral effects by inhibiting NAALADase at low concentrations. This is based on the following similarity of overlapping sequences between PBP and NAALADase I, one of several known NAALADase species, as shown below:

Active site I:

PBP: 35 AA in the 3 'direction from the N: STTK end

NAALADasa: 38 AA in the 3 'direction from the N: STQK end

Active site II:

PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motif: SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC reason: KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motif: KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC reason: ENKD

NAALADasa: 41 AA in the 3 'direction from the reason SFG: ERGV

Since beta-lactams provide their inhibition of transpeptidation of PBP from the bacterial cell wall by binding to these four active sites, it can be deduced that the similarity conserved in active site sequences and the location would confer similar binding properties of moxalactam and other compounds of β-lactam to NAALADase and possibly other neurogenic enzymes that have sequences that overlap with the four active binding site motif. This discovery coupled with the observation of the significant effects of behavior modification that result from the administration of very low doses of certain penicillin protein binding compounds has provided the opportunity for a novel approach to the prevention and treatment of conditions. of disease characterized by neurological dysfunction.

The only neurological activity profiles of the two β-lactam compounds that have been studied more widely to date, moxalactam and clavulanic acid, suggest that those compounds have activity on multiple targets of neurogenic enzymes, including NAALADase and structurally enzymes related, particularly those that could share the four active binding site motif common to both PBP and NAALADase. To identify other putative neurogenic targets for 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 II and encode the active site motif four:

1)> dbj / AP001769.2 / AP001769 Clone RP11-240F8 map 11q14 of chromosome 11 of Homo sapiens

Active site I:

PBP: 35 AA in the 3 'direction from the N: STTK end

NAALADasa: 38 AA in the 3 'direction from the N: STQK end

> dbj / AP001769: NSRK

Active site II:

PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motif: SFG

> dbj / AP001769: SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC reason: KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motif: KLG

> dbj / AP001769: KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC reason: ENKD

NAALADasa: 41 AA in the 3 'direction from the reason SFG: ERGV

> dbj / AP001769: ERSI

2)> dbj | AP000827.2 | AP000827 Clone RP of chromosome 11 of Homo sapiens.

Active site I:

PBP: 35 AA in the 3 'direction from the N: STTK end

NAALADasa: 38 AA in the 3 'direction from the N: STQK end

> dbj | AP000827.2: NSRK

Active site II:

PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motif: SFG

> dbj | AP000827.2: SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC reason: KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motif: KLG

> dbj | AP000827.2: KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC motif: ENKD 4)> gb | AC074003.2 | AC074003 Clone RP11 of chromosome 2 of Homo sapiens. Active site I: PBP: 35 AA in the 3 'direction from end N: STTK NAALADasa: 38 AA in the 3' direction from end N: STQK gb | AC074003.2 | AC074003: STQ-Active site II: PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN NAALAD Rate: 59 AA in the 3' direction from the STQK reason: SFG gb | AC074003.2 | AC074003: SFG Active site III: PBP : 111 AA in the 3 'direction from the SGC reason: KTG NAALADasa: 110 AA in the 3' direction from the SFG reason: KLG gb | AC074003.2 | AC074003: KLG Active site IV: PBP: 41 AA in the address 3 'from the SGC reason: ENKD NAALADase: 41 AA at the address 3' from the reason SFG: ERGV gb | AC074003.2 | AC074003 ER GV

NAALADasa: 41 AA in the 3 'direction from the SFG motif: ERGV> dbj | AP000827.2: ERSI 3)> dbj | AP000648.2 | AP000648 CM clone of chromosome 11 of Homo sapiens.

Active site I:

PBP: 35 AA in the 3 'direction from the N end:

STTK

NAALADasa: 38 AA in the 3 'direction from the N end:

STQK

>> dbj | AP000648.2:

NSRK

Active site II:

PBP: 57 AA in the 3 'direction from the STTK motive:

SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motive:

SFG

> dbj | AP000648.2:

SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC motive:

KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motive:

KLG

> dbj | AP000648.2:

KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC motive:

ENKD

NAALADasa: 41 AA in the 3 'direction from the SFG motive:

ERGV

> dbj | AP000648.2:

ERSI

5)> emb | AL162372.6 | AL162372 Clone RP of chromosome 13 of Homo sapiens.

Active site I:

PBP: 35 AA in the 3 'direction from the N end:

STTK

NAALADasa: 38 AA in the 3 'direction from the N end:

STQK

emb | AL162372.6:

STQ-

Active site II:

PBP: 57 AA in the 3 'direction from the STTK motive:

SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motive:

SFG

emb | AL162372.6:

SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC motive:

KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motive:

KLG

emb | AL162372.6:

KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC motive:

ENKD

NAALADasa: 41 AA in the 3 'direction from the SFG motive:

ERGV

emb | AL162372.6

ER GV

6) gb | AC024234.5 | AC024234 Clone RP1 of chromosome 11 of Homo sapiens. Active site I: PBP: 35 AA at address 3 'from end N: STTK NAALADasa: 38 AA at address 3' from end N: STQK gb | AC024234.5 | AC024234: STQ-Active site II: PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN NAALAD Rate: 59 AA in the 3' direction from the STQK reason: SFG gb | AC024234.5 | AC024234: SFG Active site III: PBP : 111 AA in the 3 'direction from the SGC reason: KTG NAALADasa: 110 AA in the 3' direction from the SFG reason: KLG gb | AC024234.5 | AC024234: KLG Active site IV: PBP: 41 AA in the address 3 'from SGC motive: ENKD NAALADasa: 41 AA at address 3' from SFG motif: ERGV gb | AC024234.5 | AC024234 ER GV

7) dbj | AP002369.1 | AP002369 Clone RP of chromosome 11 of Homo sapiens. Active site 1:

PBP: 35 AA in the 3 'direction from the N: STTK end

NAALADasa: 38 AA in the 3 'direction from the N: STQK end

dbj | AP002369.1: STQ-

Active site II:

PBP: 57 AA in the 3 'direction from the STTK reason: SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motif: SFG

dbj | AP002369.1: SFG

Active site III:

PBP: 111 AA in the 3 'direction from the SGC reason: KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motif: KLG

dbj | AP002369.1: KLG

Active site IV:

PBP: 41 AA in the 3 'direction from the SGC reason: ENKD

NAALADasa: 41 AA in the 3 'direction from the reason SFG: ERGV

dbj | AP002369.1 ER GV

The encoded protein of each of those gene sequences expressed in the brain are likely targets for behavioral and cognitive activity by β-lactams and other NAALADase inhibitors. Therefore, a procedure is provided to modify the 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 sequences

5 of genes previously identified, administer an effective amount of a β-lactam compound or other compound that can inhibit NAALADase.

In one embodiment, the NAALADase inhibitors effective for use in the various embodiments of pharmaceutical formulations and methods of the present invention, in general terms, are compounds that have selective affinity detectable by penicillin binding proteins recognized in the art which particularly include compounds containing β-lactam (hereinafter referred to as "β-lactam compounds"). Among such NAALADase inhibitor compounds, those preferred for use according to the present invention are compounds that also have good transport properties to the blood brain barrier evidenced by favorable concentration ratios of cerebrospinal fluid (CSF) / brain: serum. Furthermore, it will be appreciated that other NAALADase inhibitors recognized in the art can be used alone or in combination.

15 with penicillin protein binding compounds for the treatment and prevention of behavioral and / or cognitive disorders.

In embodiments of the invention related to pharmaceutical formulations for use in the inhibition of neurogenic NAALADase to modify behavior and / or improve cognitive function, β-lactam compounds are typically formulated in unit dosage form optionally in combination with, or as

20 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 list of United States patents: US Pat. 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.

Enhanced concentrations of active ingredients, including inhibitors of NAALADase in the brain, can also be achieved by co-administration with inhibitors of P-glycoprotein efflux such as those described in US Pat. numbers 5,889,007; 5,874,434; 5,654,304; 5,620,855; 5,643,909; and 5,591,715. Alternatively, the β-lactam antibiotic compounds useful according to the present invention, which include clavulanic acid and moxalactam, can be administered alone or in combination with β-lactamase inhibitors.

30 recognized in the art, which by themselves may or may not be β-lactam compounds or compounds that may exhibit selective affinity for penicillin binding proteins. Examples of β-lactamase inhibitors that can be used alone or in combination with other neuropeptidase inhibitors useful according to the present invention for the treatment and / or prevention of cognitive or behavioral disorders are other β-lactam compounds that may or may not exhibit activity. clinically significant independent antibacterial such

35 as clavulanic acid and thienamicin and analogs thereof, sulbactam, tazobactam, sultamicillin, and aztreonam and other monolactams.

The patent and non-patent literature is full of references describing β-lactam antibiotics, their preparation, their characterization, their formulation and their mode of action. Β-lactam antibiotics are known to present their antibiotic activity interfering with one or more biological pathways involved in the synthesis of the bacterial cell wall; more particularly, they inhibit the activity of carboxypeptidases and / or transpeptidases (or proteases) involved in crosslinking the peptidoglycan chains used as building blocks for cell wall synthesis. Therefore, β-lactam antibiotics are thought to act as inhibitors of carboxypeptidases or transpeptidases by their covalent associative binding and, by some reports, non-covalent, to one

or more than one group of such bacterial enzymes generally called penicillin binding proteins (PBP). Such enzymes serve to complete the synthesis of the bacterial cell wall by crosslinking peptidoglycan chains.

A similar peptidase-substrate interaction / inhibition according to the present invention is now suggested as a significant neurochemical pathway involved in the fundamental brain function for the cognitive and behavioral performance phenotype. Such a neurochemical mechanism is also suggested by the discovery that the administration of effective amounts of the Ala-D--Glu-Lys-D-alanyl-D-alanine peptide directly to the brain produced the same modified behavioral characteristics as those achieved by comparable concentrations of β-lactam compounds in the brain. It seems that the peptide serves as a substitute substrate for (and therefore serves to inhibit its activity) one or more neurogenic peptidases (for example, NAALADases) that normally have their activity in peptide neurotransmitters or neuromodulators, that is, NAAD, in the ordinary course of certain neurochemical processes that mediate the cognitive and behavioral performance phenotype.

Based on animal trials to date, it is believed that general classes of behavioral disorders that can be prevented or treated according to the present invention by administration of effective amounts of NAALADase inhibitors include anxiety and depression. A NAALADase inhibitor selected from those that can bind to penicillin-binding protein, for example, a β-lactam antibiotic or β-lactamase inhibitor, and / or those that exhibit inhibition of selective proteolytic activity on a bacterial protein or substrate A peptide comprising the amino acid sequence acyl-D-alanyl-D-alanine of the C-terminus, or other NAALADase inhibitor, is administered as an antiagressive agent to control impulsivity and violence in a patient suffering from autism, Tourette's syndrome, delay mental, psychosis, obsession, senile dementia or in a patient with personality disorder and a history of inappropriate aggression. In another embodiment, a deaminoglutamate analogue or an N-substituted glutamate derivative is administered in an amount effective to control impulsivity and violence in patients affected with such disease states.

Other states of neurological disease that can be treated include depression, which includes major depression (individual, recurrent, melancholic episode), atypical, dysthymia, subsindromic, agitated, delayed, co-morbid with cancer, diabetes, or post-myocardial infarction, involuntary , bipolar disorder, psychotic, endogenous and reactive depression, obsessive-compulsive disorder, or bulimia. In addition, NAALADase inhibitors can be used to treat patients suffering from pain (administered alone or in combination with morphine, codeine or dextropropoxyphene), obsessive-compulsive personality disorder, post-traumatic stress disorder, hypertension, atherosclerosis, anxiety, nervous anorexia, panic , social phobia, stuttering, sleep disorders, chronic fatigue, cognitive deficit associated with Alzheimer's disease, occasional excessive alcohol consumption, appetite disorders, weight loss, agoraphobia, memory improvement, amnesia, smoking cessation, symptoms of nicotine withdrawal syndrome, mood and / or appetite disorders associated with premenstrual syndrome, depressive mood and / or desire for carbohydrates associated with premenstrual syndrome, mood disorders, appetite disorders or disorders that contribute to recidivism associated with nicotine withdrawal, circadian rhythm disorder, border disorder izo personality, hypochondriasis, premenstrual syndrome (PMS), late luteal phase dysphoric disorder, premenstrual dysphoric disorder, trichotillomania, symptoms after suspension of other antidepressants, aggressive / intermittent explosive disorder, gambling, compulsive spending, compulsive sex, disorder use of psychoactive substances, sexual disorder, schizophrenia, premature ejaculation or selected psychiatric symptoms of stress, worry, anger, rejection sensitivity and lack of mental or physical energy.

Other examples of pathological psychological conditions that can be treated include moderate mental retardation (318.00), severe mental retardation (318.10), profound mental retardation (318.20), mental retardation of unspecified severity (319.00), autistic disorder (299.00), generalized disorder of the unspecified development (299.80), attention deficit hyperactivity disorder (314.01), behavior disorder, group type (312.20), behavior disorder, aggressive solitary type (312.00), behavior disorder, undifferentiated type (312.90), Tourette's disorder (307.23), chronic motor or vocal tics disorder (307.22), transient tics disorder (307.21), unspecified tics disorder (307.20), primary degenerative dementia of the Alzheimer type, senile onset, uncomplicated (290.00) , primary degenerative dementia of the Alzheimer type, senile onset, with confusion (290.30), primary degenerative dementia of the Alzheimer type, senile onset, with delusions tes (390.20), primary degenerative dementia of the Alzheimer type, senile onset, with depression (290.21), primary degenerative dementia of the Alzheimer type, presenile appearance, without complications (290.10), primary degenerative dementia of the Alzheimer type, presenile appearance, with confusion ( 290.11), primary degenerative dementia of the Alzheimer type, presenile appearance, with delusional ideas (290.12), primary degenerative dementia of the Alzheimer type, presenile appearance, with depression (290.13), multi-infarct dementia, without complications (290.40), multi- dementia heart attack, with confusion (290.41),

multi-infarct dementia, with delusional ideas (290.42), multi-infarct dementia, with depression (290.4 3), senile dementia not specified (290.10), unspecified presenyl dementia (290.10), alcohol withdrawal confusion (291.00), alcoholic hallucinosis (291.30), alcohol dementia associated with alcoholism (291.20), sympathomimetic poisoning by amphetamines or similar action (305.70), sympathomimetic delusional disorder by amphetamines or similar action (292.11), delusional cannabis disorder (292.11), cocaine intoxication (305.60), cocaine confusion (292.81), delusional cocaine disorder (292.11), hallucinogenic hallucinosis (305.30), hallucinogenic delusional disorder (292.11), hallucinogenic mood disorder (292.84), postalucinogenic perception disorder hallucinogens (292.89), poisoning by phencyclidine (pcp) or by arylcyclohexylamines of similar action (305.90), confusion by phencyclidine (pcp) or by ar ilyclohexylamines of similar action (292.81), delusional disorder by phencyclidine (pcp) or by arylcyclohexylamine of similar action (292.11), mood disorder by phencyclidine (pcp) or by arylcyclohexylamine of similar action (292.84), unspecified organic mental disorder by phencyclidine (pcp) or by arylcyclohexylamines of similar action (292.90), intoxication by other psychoactive or unspecified substances (305.90), confusion by other psychoactive or unspecified substances (292.81), dementia by other psychoactive or unspecified substances (292.82) , delusional disorder from other psychoactive or unspecified substances (292.11), hallucinosis from other psychoactive or unspecified substances (292.12), mood disorder from other psychoactive or unspecified substances (292.84), anxiety disorder from other psychoactive substances or not specified (292.89), personality disorder due to other psychoactive substances or unspecified (292.89), organic mental disorder not specified by other psychoactive or unspecified substances (292.90), confusion (293.00), dementia (294.10), organic delusional disorder (293.81), organic hallucinosis (293.81), mood disorder organic (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 ), unspecified anxiety disorder (300.00), body dysmorphic disorder (300.70), hypochondriasis (or hypochondriacal neurosis) (300.70), somatization disorder (300.81), undifferentiated somatoform disorder (300.70), unspecified somatoform disorder (300.70), intermittent explosive disorder (312.34), kleptomania (312.32), ludopathy (312.31), pyromania (312.33), trichotillomania (312.39) and non-impulse control disorder specified (312.39).

Additional examples of pathological psychological conditions that can be treated using protease inhibitors containing β-lactam 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 exacerbation acute (295.13), schizophrenia, disorganized, chronic with acute exacerbation (295.14), disorganized schizophrenia, in remission (295.15), schizophrenia, disorganized, unspecified (295.10), schizophrenia, paranoid, subchronic (295.31), chronic schizophrenia, paranoid (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 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), p disorder induced psychotic disorder (297.30), unspecified psychotic disorder (atypical psychosis) (298.90), bipolar disorder, mixed, severe, without psychotic characteristics (296.63), bipolar disorder, manic, severe, without psychotic characteristics (296.43), bipolar disorder, depressive , severe, without psychotic characteristics (296.53), bipolar disorder, mixed, with psychotic characteristics (296.64), bipolar disorder, manic, with psychotic characteristics (296.44), bipolar disorder, depressive, with psychotic characteristics (296.54), unspecified bipolar disorder (296.70), major depression, individual episode, with psychotic characteristics (296.24), recurrent major depression with psychotic characteristics (296.34) personality disorders, paranoids (301.00), personality disorders, schizoids (301.20), personality disorders, schizotypal (301.22), personality disorders, antisocial (301.70), personality disorders, border (301.83).

Anxiety disorders that can be treated according to the present 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 a 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) , childhood or adolescence avoidance disorder

(313.21) and hyperansitivity disorder (313.00).

Effective amounts of NAALADase inhibitors, particularly the β-lactam compounds described herein, can be used for the treatment of the following pathological psychological conditions: moderate mental retardation; severe mental retardation; deep mental retardation; autistic disorder; attention deficit hyperactivity disorder; generalized developmental disorder not specified; conduct disorder, type of group; conduct disorder, aggressive solitary type; Tourette's disorder; primary degenerative dementia of the Alzheimer type, senile onset, with confusion; primary degenerative dementia of the Alzheimer type, senile onset, with delusional ideas; primary degenerative dementia of the Alzheimer type, presenile occurrence; 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, chronic residual; 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; unspecified psychotic disorder (atypical psychosis); bipolar disorder, mixed, with psychotic characteristics; bipolar, manic disorder, with psychotic characteristics; bipolar, depressive disorder, with psychotic characteristics; unspecified bipolar disorder; major depression, individual episode, or recurrent with psychotic characteristics; personality disorders, paranoids; personality disorders, schizoids; personality disorders, schizotypal; personality disorders, antisocial; personality disorders, borderline, anxiety disorders, panic disorder, panic disorder with agoraphobia, panic disorder without agoraphobia, agoraphobia without a history of panic disorders, social phobia, simple phobia, obsessive-compulsive disorder, post-traumatic stress disorder, generalized anxiety disorder, unspecified anxiety disorder, organic anxiety disorder, psychoactive substance anxiety disorder, separation anxiety disorder, childhood or adolescence avoidance disorder and hyperansitivity disorder.

One or more neurogenic NAALADase inhibitors, which particularly include neurotropic β-lactam antibiotics or β-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; unspecified psychotic disorder (atypical psychosis); bipolar disorder, mixed, with psychotic characteristics; bipolar, manic disorder, with psychotic characteristics; bipolar, depressive disorder, with psychotic characteristics; unspecified bipolar disorder; personality disorders, paranoids; personality disorders, schizoids; personality disorders, schizotypal; personality disorders, antisocial; personality disorders, border.

Examples of psychotic conditions that are most preferably treated 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, 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; personality disorders, schizoids; and personality disorders, schizotypal.

In a preferred aspect of the present invention a treatment for anxiety is provided. Examples of anxiety disorders that are treated using the present method and pharmaceutical formulations of the present invention include anxiety disorders, panic disorder, panic disorder with agoraphobia, panic disorder without agoraphobia, agoraphobia without a history of panic disorders, social phobia simple phobia

5 obsessive-compulsive disorder, post-traumatic stress disorder, generalized anxiety disorder, unspecified anxiety disorder, organic anxiety disorder, psychoactive substance anxiety disorder, separation anxiety disorder, childhood or adolescence avoidance disorder and disorder of hyperancy.

Examples of anxiety disorders that are most preferably treated include panic disorder; phobia

10 social; simple phobia; organic anxiety disorder, obsessive-compulsive disorder; posttraumatic stress disorder; generalized anxiety disorder; and anxiety disorder not specified.

The NAALADase inhibitors used as a neurochemical functional agent in the procedures and formulations are particularly characterized by their binding to penicillin-binding proteins [as determined using the procedures described, for example, by B.G. Spratt, Properties of the penicillin-binding proteins of 15 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, Antimicrob. Agents Chemother., 23: 98-104 (1983)] and, in the case of antibiotics, by their inhibition of the activity of carboxypeptidase and / or selective transpeptidase in peptide substrates comprising the amino acid sequence Ala-D-- Glu-Lys-D-alanyl-D-alanine. Such compounds include

20 particularly β-lactam compounds.

in which

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group 25,

the chirality in C1 may be R or S, each epimer having unique biological activity; X = O;

R1 = lower alkoxy, preferably methoxy; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; Y

30 Z =

wherein R2 = lower alkyl, preferably methyl, or branched lower alkyl.

in which

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group 5, the chirality in C1 may be R or S, each epimer having unique biological activity; X = O; R1 = H; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; and Z =

wherein R2 = lower alkyl, preferably methyl, or branched lower alkyl.

15 in which

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or forming group of ester, the chirality in C1 may be R or S, each epimer having unique biological activity; X = S, S = O, SO2;

R1 = lower alkoxy, preferably methoxy;

Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; Y Z =

wherein R2 = lower alkyl, preferably methyl, or branched lower alkyl.

in which

10 V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group, the chirality in C1 may be R or S, each epimer having unique biological activity; X = S, S = O, SO2; R1 = H; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; and Z =

wherein R2 = lower alkyl, preferably methyl, or branched lower alkyl.

in which

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group 5, the chirality in C1 may be R or S, each epimer having unique biological activity; X = C; R1 = lower alkoxy, preferably methoxy; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; and Z =

wherein R2 = lower alkyl, preferably methyl, or branched lower alkyl.

15 in which W =

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or forming group of ester, the chirality in C1 may be R or S, each epimer having unique biological activity; X = C;

R1 = H; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; Y

Z =

In which R2 = lower alkyl, preferably methyl, or branched lower alkyl.

in which W =

15 V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group, the chirality in C1 may be R or S, each epimer having unique biological activity; X = C; R1 = H; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl,

pivaloyloxymethyl, acetoxymethyl, etc .; and T = Cl, F, Br, I, CH3, C2-C4 alkyl, aryl including heteroaryl, S-alkyl, S-aryl including S-heteroaryl, SO3R (R = H, alkyl, aryl), SO2R (R = H, alkyl, aryl), N-alkyl2, N-aryl2, CO2R (R = H, alkyl), P-alkyl2, P-aryl2, PO3R2 (R = H, alkyl, aryl).

in which

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group 5, the chirality in C1 may be R or S, each epimer having unique biological activity; X = C; R1 = lower alkoxy, preferably methoxy; Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl,

10 pivaloyloxymethyl, acetoxymethyl, etc .; Y

T = Cl, F, Br, I, CH3, C2-C4 alkyl, aryl including heteroaryl, S-alkyl, S-aryl including S-heteroaryl, SO3R

(R = H, alkyl, aryl), SO2R (R = H, alkyl, aryl), N-alkyl2, N-aryl2, CO2R (R = H, alkyl), P-alkyl2, P-aryl2, PO3R2

(R = H, alkyl, aryl).

15 in which W =

V = -CO2M, SO3M, SO2M, PO3M2, PO2M in which M = H or a pharmaceutically acceptable salt or ester forming group, the chirality in C1 may be R or S, each epimer having unique biological activity; X = O;

R1 = lower alkoxy, preferably methoxy;

Y = OM, M = H, a pharmaceutically acceptable salt or active ester forming group such as 1-indanyl, pivaloyloxymethyl, acetoxymethyl; Y Z =

wherein R2 = lower alkyl, preferably methyl, or branched general alkyl.

Other compounds are those compounds of formulas III, IV, III 'and IV', respectively, in which the groups W, V, X, R1, Y, M and Z are as defined for the compounds Ia-If, respectively.

Other preferred compounds, IXa-IXf; Xa - Xf, XIa - XIf, XIIa - XIIf, XI'a - XI'f and XII'a - XII'f, are those compounds of formulas IX, X, XI, XII, XI ', and XII', respectively, in which the groups W, V, X, R1, Y and M are as defined for the compounds Ia-If, respectively, and the group T is defined as for the compounds Ig and Ih, respectively.

Another group of preferred compounds XIIIa-XIIIf, XIVa-XIVf, XVa-XVf, XVIa-XVIf, XV'a-XV'f and XVI'a-XVI'f are those compounds of formulas XIII, XIV, XV, XVI, XV 'and XVI', respectively, in which the groups W, V, X, R1, Y and M are as defined for the compounds Ia-If, respectively, and the group T is defined as for the compounds Ig and Ih, respectively.

Another group of preferred compounds are compounds XVIIa-XVIIf, XVIIIa-XVIIIf, XIXa -XIXf and XXa-XXf, respectively, in which the groups W, V, X, R1, M and Y are as defined for compounds Ia - If, 10 respectively.

wherein R is hydrogen, a salt forming group or an active ester forming group; R1 is hydrogen or C1-C4 alkoxy; T is C1-C4 alkyl, halo (including chlorine, fluorine, bromine and iodine), hydroxy, O (C1-C4) alkyl or -CH2B in which B is the residue of a nucleophilic B: H, and Acyl is the Acyl OH organic acid residue.

5 Examples of such commercially available compounds (1-alkoxy-1-detia-cephemes) are moxalactam and flomoxef. Moxalactam is described and claimed in US Pat. No. 4,323,567. Moxalactam is particularly preferred due to its good transport to the blood brain barrier, thus providing a relatively high concentration ratio of that compound in the brain with respect to blood / serum levels.

In another embodiment of the invention, the moxalactam (or derivative or analog thereof) detailed for

10 parenteral administration to achieve clinically effective antibiotic tissue concentrations becomes the corresponding mono- or bis-active esters to improve oral absorption of said compounds to a level sufficient to inhibit the activity of neurogenic proteases in the brain and concomitantly effect the cognitive behavior and performance, although at an insufficient serum concentration for the clinical efficacy of the antibiotic.

Alternatively, an N-substituted glutamate derivative or a 2-deamino-2-substituted glutamate analogue is converted into a corresponding active ester derivative. Examples of suitable in vivo hydrolysable ester groups (active) include, for example, acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl, 1- (cyclohexylcarbonyloxy) prop-1-yl and (1-aminoethyl) carbonyloxymethyl; alkoxycarbonyloxyalkyl groups such as ethoxycarbonyloxymethyl and alpha-ethoxycarbonyloxyethyl; dialkylaminoalkyl groups such as ethoxycarbonyloxymethyl

20 and β-ethoxycarbonyloxyethyl; dialkylaminoalkyl groups, especially di-lower alkyl-aminoalkyl 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 phthalicyl and dimethoxyphthalidyl; and esters linked to a second β-lactam antibiotic or a β-lactamase inhibitor. An example of such chemical modification of a commercially available parenteral β-lactam antibiotic is moxalactam (Ia,

Y = OH, R1 = OCH3 and V = COM in which M = OH), is the preparation of its active ester analogue Ia in which Y = OM, M = H or an active ester, for example, 1- indanyl and V = CO2M in which M is H or an active ester in which at least one of V and Y includes an active ester moiety.

Suitable pharmaceutically acceptable salts of the carboxy group of the previously identified β-lactam antibiotics or glutamate derivatives or analogs include metal salts, for example, aluminum salts, of alkali metals such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, and substituted ammonium or ammonium salts, for example, those with lower alkyl amines such as triethylamine, hydroxyalkyl lower amines 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-efenamine, Nmethylmorpholine, N-ethylpiperidine, N-benzyl-β-phenethylamine, dehydroabyethylamine, N, N'-bisdehydroabiethydroabyethylamine

Ethylenediamine, or bases of the pyridine type such as pyridine, collidine or quinoline, or other amines that have been used to form salts with known penicillins and cephalosporins. Other useful salts include lithium salt and silver salt. The salts within the compounds of formula (I) can be prepared by exchanging salts in a conventional manner.

A penicillin or penicillin analogue of pharmaceutically acceptable salt forming or ester forming formula; R1 is H or lower alkoxy, G is hydrogen or hydroxy, and Z is amino, acylamino, CO2M, SO3M, PO3M2 or PO2M in which M is hydrogen or a pharmaceutically acceptable salt forming or ester forming group, preferably a forming group of active esters.

Penama and cepheme or sulfoxides and sulphones of non-antibiotic or weakly antibiotic cephemes and structurally related β-lactamase inhibitors such as tazobactam, clavulanic acid and sulbactam are particularly useful in applications that are important for the development of antibiotic resistance.

There is a wide variety of commercially available β-lactam antibiotics that can be used, or

be modified or formulated for use to optimize its effectiveness. Examples of such compounds are:

cephamandol naphate (Mandol)

cefazolin sodium (Zolicef, Ancef, Kefzol)

cefmetaxol sodium (Zeegazone)

cefonicide sodium (Monocid)

cefoperazone sodium (Cefobid)

ceforanide (Precef)

cefotaxime sodium (Claforan)

cefotetan sodium (Cefotan, Apatef)

cefoxitin sodium (Mefoxin)

Ceftazidime (Ceptaz, Fortaz, Tazicef, Tazidime)

ceftizoxime sodium (Kefurox, Zinacef)

Ceftriaxone (Rocephin)

ceeftriaxone sodium (Rocephin)

Cefuroxime injection (Kefurox, Zinacef)

Cefradine injection (Velosef injection)

cephalothin sodium (Keflin, Seffin)

cefapirin sodium (Cefadyl)

Moxalactam (Moxam)

amoxicillin (Amoxil, Polymox)

amoxicillin and clavulanate (Augmentin)

ampicillin (Omnipen, Principen)

ampicillin and sulbactam (Unasyn)

azlocillin (Azlin)

bacampicillin (Spectrobid)

carbenicillin (Geocillin, Geopen)

Cefaclor (Ceclor)

cefadroxil (Duricef, Ultracef)

Cefamandol (Mandol)

cefazolin sodium (Ancef, Kefzol)

Cefixin (Suprax)

cefmetazole sodium (Zefazone)

cefonicide (Monocid) cefoperazone (Cefobid) ceforanide (Precef) cefotaxime (Claaforan) Cefotetan (Cefotan) cefoxitin (Mefoxin) cefprozil injection oral cefprozil (Cefzil) Ceftazidime (Ceptaz, Fortax) ceftizoxime (Cefizox) Ceftriaxone (Rocephin) Cefuroxime (Ceftin, Zinacef) cephalexin (Keflex, Keftab) cephalothin (Keflin) cefapirin (Cefadyl) Cefradine (Anspor, Velosef) Cloxacillin (Cloxapen, Tegopen) cyclacillin Dichloxacillin (Dycill, Pathocil) loracarbef injection oral loracarbef methicillin (Staphcillin) mezlocillin (Mezlin) Moxalactam (Moxam) Nafcillin (Nafcil, Unipen) oxacillin (Bactocill, Prostaphlin) penicillamine (Cuprimine, Depen) Penicillin G (Wycillin, Pentids, Bicillin LA) penicillin V (Veetids, V-Cillin K) piperacillin (Pipracil) Ticarcillin (Ticar) Ticarcillin and Clavulanate (Timentin) Other commercially available β-lactam antibiotics include imipenem (primaxin) that is administered

normally in combination with cilastatin (which prevents any degradation by renal enzymes). Others

β-lactam antibiotics include meropenem (Meronem / Zeneca) and aztreonam (Azacetam). Other penis include

biapenem, panipenem, carumonam and ritipenam. Β-lactamase inhibitors are particularly useful

recognized in the art that also have cross-inhibition of NAALADase, and that may or may not present

antibiotic independent activity.

Those β-lactam antibiotic compounds detailed for parental administration can be modified, as indicated above, as their active ester derivatives to improve oral absorption in order to reach levels in the brain of the compound sufficient to inhibit neurogenic protease, but not necessarily sufficient to provide clinically effective levels in antibiotic blood. Other antibiotics that can be based for their antibiotic efficacy on inhibition of protease-mediated cell wall synthesis, for example, gamma-lactones and / or quinoline antibiotics, can also be used.

Other compounds capable of inhibiting neurogenic peptidases by the proposed mechanism for the activity of β-lactam compounds can be identified using molecular discovery techniques recognized in the art, for example, those described in US Pat. 5,552,543. That patent described algorithms to detect the correlation between antibacterial activity and the "low key" interactions that take place between penicillin-binding proteins and β-lactam antibiotics. Such molecular modeling techniques may be related to other drug modeling techniques (for example, those described in published PCT International Publication No. WO 99/10522) to identify compounds with good transport efficiencies to the blood brain barrier to identify optimally effective compounds for use Therefore, for treatments according to the present invention that choose as neurogenic NAALADase target it is not only important that compounds useful in the present invention be active as inhibitors of the neurological protease chosen as the target (NAALADase), but it is also important that such compounds can administered with some degree of threshold efficiency across the blood brain barrier to provide effective protease inhibition concentration of the drug in the brain. Such transport properties to the blood brain barrier can be inherently derived from the structure of the compound, or such compounds can be formulated and / or conjugated with other chemical entities effective to enhance transport to the blood brain barrier. There has been a significant research and development effort aimed at the preparation and formulation of compounds to enhance their transport to the blood brain barrier, and such technologies can be applied to enhance the concentration in the brain of protease inhibitors and adjuvants for the same useful according to the present invention.

Animal tests indicate an effective threshold dose of moxalactam (parenterally administered) that is approximately 50 µg / kg body weight. Based on data from animal tests and the known distribution of parenterally administered moxalactam between the brain and other body tissues, the minimum effective neurogenic protease inhibitor concentration of moxalactam in the brain is approximately 30 nM. Clavulanic acid has been shown to be an effective neurogenic NAALADase inhibitor when i.p. at less than 1 microgram per kilogram of body weight. The range of effective dosage levels of NAALADase inhibitors when used in the treatment of behavioral and / or cognitive disorders according to the present invention will depend significantly on the patient's body weight, the affinity of the inhibitor for the target neurogenic protease, the characteristics of transport to the blood-brain barrier of the active compound, the mode of administration and the optional use of available drug formulation / conjugation technologies available for enhancing transport to the blood-brain barrier. For parenterally administered moxalactam, the minimum effective dose in hamsters and other test species is approximately 50 micrograms per kg body weight, more or less. It is estimated that the use of moxalactam in an oral pharmaceutical form, preferably modified or derivatized in the form of an active ester, ranges from about 2.5 to about 50 mg per dose, much lower than the dose of moxalactam necessary to provide antibiotic concentration. therapeutically effective. The effective oral dose of clavulanate is expected to be about 0.1 to about 10 mg per dose. Clavulanate is absorbed orally and has good transport to the blood brain barrier.

The effective doses of other protease inhibitors will again vary, depending on their inherent affinity for the target protease, the route of administration selected, the weight of the patient and the efficiency of transport to the blood brain barrier. The effective dosages of NAALADase inhibitors used according to the present invention can be readily determined empirically using animal models coupled to the use of recognized analytical techniques in the art. Normally, the dosage levels for the β-lactam antibiotic compounds used in the methods and formulations of the present invention are lower than those necessary to achieve clinically effective antibacterial levels. Parenteral dosages of βlactam antibiotic compounds may 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 according to the present invention when the patient's circumstances dictate that in the judgment of the attached physician. Therefore, for example, if the patient / clinical conditions are such that the inherent antibiotic activity of β-lactam compounds is not considered to be a complicated contraindication, larger doses of the antibiotic may be used up to or exceeding dosage levels. which can provide clinically effective levels in antibiotic threshold blood to treat patients in need of therapy effected by NAALADase inhibition according to the present invention.

Other NAALADase inhibitors recognized in the art that can cross the blood brain barrier in effective amounts can be used for the treatment of behavioral and cognitive disorders. For example, they can be used to improve cognitive performance in patients suffering from dementia or to reduce aggression. Examples of known NAALADase inhibitors include general metallopeptidase inhibitors such as O-phenanthroline, metal chelators such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol ether bis (betaminoethyl) -N acid, N-tetraacetic acid (EGTA) and peptide analogs such as cuesqualic acid, aspartatoglutamate (Asp-Glu), Glu-Glu, Gly-Glu, -Glu-Glu and beta-N-acetyl-L-aspartate-L-glutamate. Other NAALADase inhibitors are the most recently described compounds of formula

wherein X is RP (O) (OH) CH2- [see US Pat. No. 5,968,915]; RP (O) (OH) NH- [see patent for

5 USA No. 5,863,536]; RP (O) (OH) O- [see US Pat. No. 5,795,877]; RN (OH) C (O) Y- or RC (O) NH (OH) Y in which Y is CR1R2, NR3 or O [see US Pat. No. 5,962,521]; or X is RS (O) Y, RSO2Y, or RS (O) (NH) Y in which Y is CR1R2, NR3 or O [see US Pat. No. 5,902,817].

Each of the US patents. Above mentioned suggests uses of the NAALADase inhibitors described in the treatment of disease states associated with glutamate abnormality that include epilepsy, stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, schizophrenia ; chronic pain, ischemia and neuronal injury. The discovery underlying the present invention allows the use of penicillin-binding protein (carboxypeptidase inhibitors)

or bacterial transpeptidase), particularly β-lactam antibiotics and β-lactamase inhibitors to develop therapeutic protocols for such disease states based on previously non-activity

15 appreciated of such compounds as NAALADase inhibitors. More recently, high concentrations of glutamate in nerve tissues have been associated with multiple sclerosis, and it is contemplated that inhibition of NAALADase in such tissues and, consequently, inhibition of their glutamate production may provide therapeutic benefit to patients afflicted with the disease. either reducing the severity of symptoms or reducing the frequency of their appearance.

The present invention further provides certain pharmaceutical formulations for the treatment of behavioral or cognitive disorders and other disease states associated with the production of abnormal concentrations of glutamate in nerve tissues and other tissues that harbor NAALADase activity as defined in claim 13 Generally, the formulation comprises a neurologically active principle that includes a compound that can inhibit a bacterial enzyme and that can inhibit a neurogenic peptidase (e.g.,

NAALADase) which is known, by empirical evidence, that acts selectively on a peptide comprising the amino acid sequence Ala-D--Glu-Lys-D-alanyl-D-alanine, and a pharmaceutically acceptable carrier thereof. In one embodiment, the pharmaceutical formulation in a unit dosage form comprises an amount of a β-lactam compound that can inhibit NAALADase activity in a patient who experiences or is predisposed to develop a medical condition that could be prevented or treated to reduce its symptom

30 by inhibition of NAALADase. The amount of peptidase inhibitor (NAALADase) and the nature of the vehicle depend, of course, on the intended route of administration. The amount of inhibitor is the amount effective to provide after administration by the predetermined route of administration a concentration of the inhibitor in the tissue in which inhibition of NAALADase is desired, for example, in the brain effective to treat and reduce symptoms of behavioral or cognitive disorders chosen as a target or other disorders that may

35 be treated by inhibition of NAALADase activity. In embodiments using β-lactam antibiotic compounds, the amount of peptidases inhibitor in the present formulations is normally less than that which can provide clinically effective inhibition of bacterial proteases, that is, less than that which can provide antibiotically effective levels when administered to a patient in the pharmaceutical form provided. Peptidase inhibitors for use according to the present invention can be combined with one

40 or more pharmaceutically acceptable carriers, and can be administered, for example, orally in forms such as tablets, capsules, oblong tablets, dispersible powders, granules, lozenges, mucosal patches, sachets and the like. The NAALADase inhibitor may be combined with a pharmaceutically acceptable carrier, for example, starch, lactose or trehalose, alone or in combination with one or more excipients that form tablets and pressed into tablets or lozenges. Optionally such

Tablets, oblong tablets or capsules can be enteric coated to minimize hydrolysis / degradation in the stomach. Oral pharmaceutical formulations contain about 1 to about 99% by weight of active ingredient and about 1 to about 99% of a pharmaceutically acceptable carrier and / or formulation excipients. Optionally, when β-lactam antibiotics are used as NAALADase inhibitors, the pharmaceutical forms can be formulated by combining them with

50 a P-glycoprotein inhibitor to provide an enhanced half-life of the drug and brain concentrations of the active substance. Alternatively, the protease inhibitor can simply be co-administered with a P-glycoprotein or β-lactamase inhibitor; or the pharmaceutical form may comprise a β-lactamase inhibitor (itself is also a NAALADase inhibitor) alone or in combination with a P-glycoprotein and a carrier.

In another embodiment of the invention, the pharmaceutical preparations may contain, for example, from about 2.5% to about 90% of the active ingredient in combination with the vehicle, more usually between about 5% and about 60% by weight of active substance. The pharmaceutical formulations according to an embodiment of the present invention are formulated for oral administration, that is, administration by oral ingestion or oral or sublingual administration (in the form of sachets, lozenges and / or patches for the oral mucosa). In another embodiment, the pharmaceutical form is formulated for oral administration in a prolonged release pharmaceutical form formulated to release the active ingredient for a predetermined period of time.

Also within the scope of the present invention are topical pharmaceutical forms that include transdermal patches, intranasal formulations and suppository unit dosage forms containing the active protease inhibitor and excipients, adjuvants and conventional non-toxic pharmaceutically acceptable carriers adapted for such routes of administration. .

Pharmaceutical formulations according to the present invention may alternatively be administered by parenteral routes of administration including subcutaneous administration, intraperitoneal administration, intramuscular administration and intravenous administration. Such parenteral pharmaceutical forms are normally in the form of aqueous solutions or dispersions using a pharmaceutically acceptable carrier such as isotonic saline solution, 5% glucose or other well-known pharmaceutically acceptable liquid excipient composition.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile or lyophilized powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form must be sterile and must be stable under the conditions of preparation and storage, and must be preserved against the contaminating action of microorganisms. The vehicle for injectable formulations may be a solvent or dispersion medium containing, for example, water, ethanol or a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof and vegetable oil.

Parenteral pharmaceutical forms of peptidase inhibitors useful for the treatment of behavioral and cognitive disorders and other disease states sensitive to inhibition of neurogenic peptidases can also be formulated as injectable prolonged-release formulations in which the protease inhibitor is combined with one or more natural or biodegradable or biodispersible synthetic polymers such as carbohydrates including starches, gums and etherified or esterified cellulosic derivatives, polyethers, polyesters (particularly polylactide, polyglycolide or polylactide 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 molded polymer matrix implants that are well known in the art for their function as "drug delivery systems of" slow release type ”that provide extended release of biologically active components. Such compositions can be prepared using formulation techniques recognized in the art and designed for any of a wide variety of drug release profiles.

The administration of pharmaceutical compositions for use in the present invention may 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 a day that the pharmaceutical formulation is administered may vary depending on the condition of the patient and the environment. The level of efficacy and optimal dosage and the pharmaceutical form for any protease inhibitor administered for use within the scope of the present invention depends on the patient and is adjustable within reasonable intervals in the judgment of the attached physician. The formulation is normally administered for a period of time sufficient to treat or prevent the disease state of the patient, for example, to modify the behavioral or cognitive performance of the patient being treated. Protease inhibitor formulations can continue to be administered using the same or attenuated dosing protocol for the prophylaxis of the disease state chosen as the target.

With knowledge of the existence and function of a neurogenic protease or proteases, such a compound or compounds can be easily separated from brain homogenates, cerebrospinal fluid (CSF) or brain tissue extracts and purified for use in elucidating structures and in Drug discovery applications. Therefore, using techniques recognized in the art that specifically include affinity chromatography and / or high pressure liquid chromatography, one or more neurogenic proteases that have a definite neurochemical function determining the behavioral phenotype and cognitive performance can be isolated and characterized. Thus, for example, a β-lactam antibiotic can covalently bind, optionally via a cleavable linker, to a solid substrate, for example, magnetic beads or functionalized silica to form a solid phase capable of selective affinity / interaction with the (s) ) Protease target (s). The solid substrate carrying the β-lactam compound is then contacted with CSF, brain homogenate or brain extract at one or more pH levels for a period of time sufficient to allow the reaction (or associative binding) of the neurogenic protease to the immobilized β-lactam compound. Alternatively, a protein or peptide substrate comprising the acyl-D-alanyl-D-alanine amino acid sequence can be immobilized on the solid substrate and used as an affinity probe for the target neurogenic protease.

The covalently or associatively bound protease can then be separated from the extract and then released from the solid substrate by a technique dependent on the nature of the immobilized protease-probe interaction. If the protease forms a covalent bond with the immobilized β-lactam compound, the resulting complex can be released from the solid phase substrate, for example, by cleaving the cleavable linker to provide a neurogenic protease, more particularly a derivative thereof, substantially free. of non-protease components. Alternatively, and preferably, the probe used for the protease affinity column has associative (non-covalent) affinity selected by the protease so that the protease can be subsequently released from the solid phase by changing the stringency (ionic strength or pH of the liquid phase ) to release the protease from the solid in a form substantially free of non-protease components.

Another method for enabling and identifying the neuroprotease target of the methods and pharmaceutical formulations of the present invention involves using a penicillin or cephalosporin labeled with a radionuclide (H3). A solution of that is injected into the brain of a test animal and after a predetermined period of time the animal is euthanized and the brain is immediately extracted / homogenized. The brain extract / homogenate is then subjected to one or more separation procedures such as centrifugation, dialysis and chromatography (eg, gel filtration chromatography). Separate chemical species are analyzed for the presence of the brand, and the labeled species are isolated, optionally subjected to additional purification procedures, and subjected to physical-chemical analysis for elucidation of the structure.

Using analytical techniques / amino acid sequencing of proteins recognized in the art, the structure of neurogenic proteases can be easily elucidated thus allowing the preparation of nucleic acid probes encoding at least a part of the protease. The probes can then be used to recover DNA encoding the protease from available gene libraries. Gene constructs chosen as the target are analyzed and cloned using cloning techniques recognized in the art to provide engineered, eukaryotic or prokaryotic microorganisms, which can be used to express unlimited amounts of pure protease that can then be used in diagnostic applications. or drug screening.

In a related embodiment, the protease is conjugated in the form of a hapten to inoculate murine or other species for the production of monoclonal antibodies that recognize one or more epitopes of the neurogenic protease. Antibody production can be carried out using hybridoma formation techniques recognized in the art well known to those skilled in the art. Such procedures are summarized, particularly with respect to the formation of monoclonal antibodies that have specificity for collagen proteins, in US Pat. 4,628,077.

The antibodies may themselves be conjugated by covalent bond or affinity bond (such as by a biotin-avidin complex) with a detectable label, for example, a radionuclide, an enzyme or a fluorescent or electroluminescent species. Such labeled antibodies can be used in diagnostic kits and procedures to analyze, for example, cerebrospinal fluid samples, to determine a patient's behavioral or cognitive status based on neuroprotease concentrations. Such diagnostic tests can also be used to assess the patient's response to pharmacotherapy and to monitor the patient's condition, again based on the concentration of neurogenic protease. The process can be carried out using competitive specific or sandwich-type binding assays recognized in the art well known to those skilled in the art.

Therefore, according to yet another embodiment, a method for assessing the neurochemical state of a patient is disclosed, comprising the step of contacting a sample of cerebrospinal fluid with a labeled antibody that can specifically bind to an epitope of the responsible neurogenic protease or proteases. of the modulation of neurochemical pathways determining the level of cognitive performance and / or phenotype of the patient's behavior, and finally comparing the levels detected with those of patient populations of predetermined cognitive or behavioral phenotypes.

In yet another embodiment a peptide neuromodulator or neurotransmitter is disclosed in substantially pure form. The neuropeptide is characterized by its mediation in the patient's behavior and cognitive abilities, and additionally by its function as a substrate for neurogenic protease, which can itself be inhibited by both the moxalactam D-isomer as a peptide comprising the acyl sequence. D-Alanyl-D-Alanine. The neurogenic protease that exhibits selective activity on the peptide neuromodulator or neurotractor may have proteolytic activity comprising transprotease or carboxyprotease activity (or alternatively exposed transpeptidase or carboxypeptidase activity). The neuropeptide can be isolated from cerebrospinal fluid or brain homogenate using a procedure similar to that described above for the isolation and purification of neurogenic protease. However, instead of using a solid substrate with covalently bound β-lactam antibiotic as an affinity probe for the target molecule, penicillin-binding protein (or an optionally chemically modified form of the isolated neurogenic protease itself) is immobilizes on the surface of the solid substrate. Penicillin-binding protein (PBP) exhibits selective affinity for the peptide neuromodulator / neurotransmitter, and the peptide target molecules preferentially bind to the solid substrate when the solid phase carrying PBP is contacted with brain homogenate or cerebrospinal fluid under a or more alternative conditions of ionic strength and / or pH. The solid phase carrying the PBP can be used in the form of a chromatographic column, that is, in an affinity chromatography application, or the solid substrate can simply be dispersed in a homogenate or brain or cerebrospinal fluid extract to selectively bind to the neuromodulator / Peptide neurotransmitter that can then be released from penicillin-binding protein, isolated and further purified, if necessary, by high pressure liquid chromatography to produce said peptide neuromodulator / neurotransmitter in substantially pure form free of other proteinaceous compositions. The peptide neurotransmitter can itself be used as a neurologically active active ingredient or it can be used as a serial head for drugs for the development of related neuroactive active ingredients. In addition, the structure of the purified neurotransmitter can be easily determined by analysis of amino acid sequences recognized in the art that will allow the skilled physician to prepare a nucleic acid probe (as possibly a synthetic gene) to isolate DNA sequences encoding the neurotransmitter / neuromodulator. peptide Finally, similar to the use of the gene for the neurogenic protease described above, the isolated gene construct can be cloned and used in the production of clinical quantities of the peptide neurotransmitter / neuromodulator for drug intervention, for the production of antibodies or in discovery protocols. of drugs

In a further aspect it is contemplated that a patient suffering from a cognitive or behavioral disorder can be vaccinated using a vaccine prepared, for example, as a conjugated neurogenic protease that can elicit an antibody response in the inoculation of the patient sufficient to attenuate the function of the neurogenic protease and concomitantly modify the behavioral and / or cognitive phenotype of the patient to improve other conditions of patients associated with abnormal glutamate concentration / activity that include, but are not limited to, those discussed earlier in this document. In one embodiment, the conjugate comprises a haptane conjugate of one or more peptides comprising a sequence of 4 to 20 amino acids of NAALADase comprising one or more of the active site motifs of NAALADase or PBP, as described above.

The above-described embodiments of the present invention are derived in part from the mechanism of action deduced from the data collected in cognitive models of behavior and animal skills described below. Other embodiments of the invention will be apparent from the analysis of the data obtained in the following experimental examples, but which are illustrative of the behavior modification and cognitive performance and the improvement that can be obtained by the use of the procedure and the formulations of the present invention

Experimental examples

Marketed in 1981-1982, moxalactam (Mox) was widely used worldwide as an antibiotic similar to third-generation cephalosporins. Clinical efficacy and safety were evaluated in more than 2,200 patients with bacterial infections (Jackson et al. 1986). Of the 260 patients treated with Mox for gram-negative meningitis, 241 (93%) showed satisfactory response to antibiotic therapy. Patients were treated with 4 g of Mox every 8 h for 2-3 weeks. Peak plasma concentrations occur within one h after IM injection with a elimination half-life of 2.3 h. There is no accumulation with multiple injections that occur at intervals of 8-12 h. Moxalactam can penetrate the blood brain barrier. The cerebrospinal fluid (CSF) levels of Mox range from 25-39 µg / ml after an IV dose of 2.0 g of drug. It is estimated that the concentration in CSF as a percentage of serum concentration is 20%. Isomer D has antibacterial activity and has a fraction that does not bind to plasma protein greater than the isomer

L.

STUDIES OF BEHAVIOR WITH MOXALACTAMA

PROCEDURES

Animal Care

Golden Syrian male hamsters (Mesocricetus auratus) (140-150 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, IN) were individually housed in Plexiglas cages (24 cm x 24 cm x 20 cm), kept in a reverse cycle of light: darkness (14L: 10O; the lights come on at 7:00 p.m.) and food and water were provided at will. The animals acclimatized to the reverse cycle of light: darkness for at least two weeks before being tested. All behavioral tests were performed during the dark phase of the circadian cycle. All animals were purchased and cared for according to the guidelines published in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 85-23, revised in 1985).

Offensive aggression

Agonist behavior can be classified as both offensive and defensive aggression (Blanchard and Blanchard, 1977; Adams, 19798; Albert and Walsh, 1984). Offensive aggression is characterized by the aggressor who initiates an attack on an opponent, while defensive aggression lacks active focus. Both types of aggression have their own unique neurobehavior systems. The stimuli that provoke the offensive and defense attack are different, since they are the sequences of behaviors that accompany each agonist response. While many of the empirical data that support the idea 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 golden male hamsters tested in a resident / intruder paradigm, an established model of offensive aggression (Ferris and Potegal 1988). Placing a strange male hamster in the home cage of another male hamster causes a well-defined sequence of resident agonist behaviors that include offensive aggression.

Measures and behavior analysis

Hamsters are nocturnal and as such all behavioral tests were performed during the first four hours of the dark phase under dim red lighting. The resident was scored for offensive aggression, for example, latency to bite the intruder, the total number of bites, total contact time with the intruder and the marking of slits during a 10-minute trial period (Ferris and Potegal, 1988) . Ijada marking is an olfactory communication form in which a hamster arches its back and rubs the glands of the pheromone-producing ijada against objects in the environment (Johnson, 1986). The frequency of tagging is greatly enhanced during aggressive encounters and is particularly strong in dominant animals that start and win fights (Ferris et al., 1987).

Parametric data, i.e. latencies and contact time, were analyzed with a unilateral ANOVA followed by Newman-Keuls post-test. Non-parametric data, that is, the number of bites and slit markings, were analyzed with Kruskal-Wallis tests followed by Mann-Whitney U tests to determine differences between groups. Two sample comparisons were analyzed with t-tests for paired and independent data for parametric data and Wilcoxon and Mann-Whitney tests for paired and independent non-parametric data, respectively.

RESULTS

I. High dose moxalactam

In a pilot study, Mox (50 mg / kg in a volume of approximately 150 µl) was administered intraperitoneally (PI) to six male hamsters previously screened for aggressive behavior towards smaller intruders. The treatments with Mox and saline vehicle were compensated so that each animal received both treatments separated by at least 48 h. The animals were tested 90 min after the estimated treatment period to reflect peak plasma levels of Mox (Jackson et al. 1986). Moxalactam was dissolved in 0.9% NaCI and stored on ice. Fresh was prepared for each study.

Resident animals treated with saline vehicle bit the intruders in less than a min (Fig. 1). After treatment with Mox, the average latency to bite increased to more than six min (p <0.05). In addition, the number of bites during the 10-minute observation period was significantly reduced (p <0.05). However, the contact time, that is, the time the resident spends in smelling and exploring the intruder was also significantly reduced (p <0.01). The decrease in the marking of slits did not reach significance, but there was a trend (p <0.07).

Summary

The general decrease in all behavioral measures associated with offensive aggression increases the possibility that the dose of 50 mg / kg of Mox has non-specific depressive effects on motor activity and activation. To examine this possibility, dose response studies were necessary to find the lowest dose of Mox that effectively inhibited offensive aggression without altering other behaviors.

II. Moxalactam dose response

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 µg / kg) was tested in six animals (Figs. 2 and 3 ). The treatments were compensated by receiving each animal each separate treatment for at least 48 h. The latency to bite was significantly different between treatments (F (5.30) = 5.66; p <0.001). Moxalactam treatment with a dose of 5.0 µg and less had no effect on any of the behavioral measures of offensive aggression. However, the dose of 50 µg / kg significantly delayed latency to bite more than seven min (p <0.001) with respect to vehicle control. Doses of 500 µg and 5.0 mg also significantly increased latency to bite. As expected, the same dose that increased the latency to bite also decreased the number of bites (H = 24.12; p <0.001). Animals treated with 50 µg of Mox showed a significant reduction in bites (p <0.05). In fact, three of the six animals never bit at all in the 10-minute observation period. The contact time was significantly different between treatments (F (5.30) = 2.5; p <0.05). Doses of 500 µg and 5 mg significantly reduced contact time with respect to vehicle control (p <0.05 and p <0.01, respectively). Offset marking was not significantly different between groups (H = 9.256; p <0.09).

Summary

These data identify the dose of 50 µg / kg of Mox as very effective in inhibiting offensive aggression without significantly reducing the contact time and the marking of slits. Larger doses of Mox, while being effective in reducing aggression measures, also reduced contact time. Hence, 50 µg doses appear to be better for future behavioral tests. Having identified the most effective dose of Mox, a deeper study was needed using a larger number of animals and a wider spectrum of behavioral tests.

III. Behavioral tests with 50 µg of moxalactam

Offensive aggression

Thirteen hamsters were tested for offensive aggression after treatment with saline vehicle or 50 µg / kg of Mox (Fig. 4). Both treatments were administered IP in a volume of approximately 150 µl. The animals were tested 90 min after injection. Each animal received both treatments. The order of injections was compensated with no less than 48 hours between treatments. Moxalactam significantly increased latency to bite (p <0.001) and reduced the number of bites (p <0.01). There was no significant change in the contact time or the marking of slits.

Summary

This larger study of low dose Mox corroborates the dose response study that confirms that Mox can effectively reduce offensive aggression without altering social behavior as measured by time spent with the intruder.

Motor activity in an open field

Six animals were tested for general motor activity in an "open field" after treatment with saline vehicle or 50 µg / kg of Mox (Fig. 5). The study was compensated by receiving each animal each treatment. Ninety minutes after the injection, a single animal was placed in a large clean Plexiglas cage (48 x 32 x 40 cm) lacking any bed of straw. This open field was delineated in equal quadrants by adhesive tape over the bottom of the cage. The animals were scored for motor activity counting the number of quadrants crossed in 1 min. There was no significant difference between treatments in the open field activity.

Olfactory discrimination

Sixteen animals were treated with vehicle or 50 µg / kg of Mox and tested for olfactory discrimination by measuring their latency to find hidden sunflower seeds (Fig. 5). Injections were compensated by receiving each animal each treatment. Before the test, the animals fasted for 24 h. Ninety minutes after the injection the animals were briefly removed from their home cage while six sunflower seeds were buried under the straw bed in a corner. The animals were placed back in their home cage and scored for latency to find the pipes in an observation period of 10 min. The latency to find the pipe was significantly (p <0.001) reduced in animals treated with Mox with respect to vehicle controls. Surprisingly, all the pipes were quickly consumed in less than five minutes after treatment with Mox, but not with saline. In reality, none of the sixteen animals consumed all the pipes after saline with respect to all the animals treated with Mox.

Sexual activity

Six animals were tested during a five-minute observation period for sexual activity after treatment with saline vehicle or 50 µg / kg of Mox (Fig. 6). The study was compensated by receiving each animal each treatment. Ninety minutes after the injection, the animals were scored for latency to copulate and the number of intrusions, that is, coupling attacks, towards a receptive female located in their home cage. Female golden hamsters were ovariectomized under general anesthesia. After recovery, the animals were treated with a single 50 mg SC injection of estradiol benzoate for three consecutive days to induce sexual receptivity. On the day of the test, females sensitized with estrogens were introduced into the home cage of the experimental males. The first investigation by the males routinely produced robust lordosis in the female. Lordosis is a stereotyped posture characterized by intense sustained vertebral dorsal flexion.

After vehicle treatment, the animals copulated and pushed a receptive female in approximately 30 s. The time to copulate increased significantly (p <0.05) after treatment with Mox. Although both treatments showed a high attack of coupling, animals treated with Mox showed a tendency towards a reduced intrusion rate (p <0.07).

Summary

Moxalactam seems to have a very good serenic profile. Serénicos are drugs used to treat impulsivity and violence (Olivier and Mos, 1991). Serénicos should suppress offensive aggression without interfering with social, appetite and cognitive behaviors. The social interest in an intruder, that is, the contact time, is not altered by Mox. Offset marking and activity in an open field also remain unchanged with drug treatment, evidence that activation and motor activity is normal. Fasting animals treated with Mox may find hidden sunflower seeds better, evidence that drug treatment does not interfere with smell or motivation to find food; Actually, you can boost it. Interestingly, the Mox treatment reduced the latency to copulate with a receptive female and decreased, although not significantly, the coupling attacks in a five-minute observation period. It should be noted that the animals treated with Mox were still very sexually active, except that the behavior seemed less intense. This anti-aggressive effect of Mox combined with a placating sexual activity could have therapeutic value in the treatment of violent sex offenders.

The 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 this possibility it was necessary to test Mox in a model used to screen drugs for their effect on anxiety

IV. Moxalactam test for anxiolytic activity

Labyrinth in raised cross

The elevated cross labyrinth was developed for the detection of anxiolytic and angiogenic drug effects in the rat (Pellow et al., 1985). The procedure has been validated in terms of behavior, physiologically and pharmacologically. The cross labyrinth consists of two open arms and two closed arms. Rats will naturally make fewer entries in open arms than in closed arms and spend significantly less time in open arms. Confinement in open arms is associated with behavior related to significantly more anxiety and higher levels of stress hormone than confinement in closed arms. Clinically effective anxiolytics, for example, chlordiazepoxide or diazepam, significantly increase the percentage of time spent in open arms and the number of entries in open arms. In contrast, anxiogenic compounds such as yohimbine or amphetamines reduce the entries in the open arms and the time spent in the open arms.

Process

Male Wistar rats weighing 250-300 g were housed in a group in a normal 12:12 light-dark cycle with the light on at 08:00 h and food and water were provided at will. The cross labyrinth consisted of two open arms, 50 x 10 cm, and two closed arms 50 x 10 x 40 cm with an open roof, arranged so that the two open arms were opposite each other. The labyrinth rose to a height of 50 cm.

Eight animals were tested in the cross labyrinth 90 min after IP injection with 50 µg / kg of Mox and saline vehicle. The order of the treatments was compensated with at least 48 hours between injections. At the beginning of the experiment, the animal was placed in the center of the cross labyrinth oriented towards the closed arm. During an observation period of five minutes the animals were scored for latency to enter the closed arm, time spent in the closed arm and the number of entries in the open arms after the first occupation of the closed arm (Fig. 7). Mox treatment significantly increased the latency to enter the closed arm (p <0.05) with respect to the vehicle. The time spent in the closed arm was significantly reduced (p <0.01), while the number of entries in the open arms was significantly high (p <0.05).

Summary

These data show that Mox administered at a dose of 50 µg / kg has anxiolytic activity. This finding enhances the Moen's serenic profile and delineates it from previous serenics such as eltoprazine that suppressed offensive aggression, increasing, in part, fear and anxiety. These data also demonstrate that Mox may have therapeutic value as an anxiolytic.

However, Mox's anxiolytic activity raises other questions about behavior specificity. Many anxiolytics, particularly benzodiazepines, are sedatives and can reduce overall motor activity and can also act as amnesics and interfere with learning and memory. As it was shown that the Mox had no effect of marking of slits or activity in an open field it is unlikely to act as a general sedative. However, it was necessary to test the Mox for any adverse effects on learning and memory.

V. Moxalactam test for anxiolytic activity

Moxalactam vs. Chlordiazepoxide

Procedures

Because Mox and CDP have different bioavailability profiles, for example, brain penetration, their activity in the CNS could not be compared by administering systemic injections of equimolar concentrations of each drug. Instead, it was necessary to administer both drugs directly into the cerebroventricular system to bypass the blood brain barrier. The animals were anesthetized with sodium pentobarbital (50 mg / kg), guide cannulas for microinjection directed to the lateral ventricle were implanted and allowed to recover for two days before being tested. Groups of six animals were each tested with Mox or CDP. Each animal received a drug injection and 0.9% NaCl vehicle on two separate days. The order of the injections was compensated and separated two days. Both Mox and CDP were prepared in 0.9% NaCl at a concentration of 1 mM. All injections were administered in a volume of 2 ul for 10 s in fully conscious animals enclosed. Sixty min later, the animals were tested in the cross labyrinth during an observation period of 3 min and scored for behaviors as previously noted.

Results

Mox treatment significantly delayed (p <0.05) the time it took to enter the closed arm with respect to vehicle treatment (Fig. 17). The treatment with Mox caused the animals to spend most of their time in the arms with light from the cross labyrinth. The time spent in the dark was significantly (p <0.01) shorter after treatment with Mox with respect to vehicle. The treatment of CDP at 1 mM concentration had no effect either on the latency to enter the closed arm nor on the time spent in the closed arm with respect to the vehicle treatment.

Control of non-specific depression of motor activity

When the CDP is administered systemically to rodents in doses of 5-15 mg / kg it is a sedative and reduces motor activity. However, this reduction in motor activity disappears after repeated administration of CDP for several days. Only after insensitizing the animals for the motor effects of the CDP are they tested in the cross labyrinth for anxiolytic activity. To control any non-specific effect of Mox and CDP on motor activity after direct injection into the brain, the animals were tested in the open field 30 min before being tested on the cross (Fig. 18). There was no significant effect of any anxiolytic on general motor activity.

Summary

The finding that Mox is an anxiolytic potentiates its serenic profile and delineates it from previous serenics such as eltoprazine that suppressed offensive aggression, partly increasing fear and anxiety. On an equimolar basis, Mox showed anxiolytic activity administered directly in the brain with respect to the CDP that did not have any. These data show that Mox can have therapeutic value as an anxiolytic, as well as as a serenic.

However, Mox's anxiolytic activity raises other questions about behavior specificity. Many anxiolytics, particularly benzodiazepines, are sedatives and can reduce overall motor activity and can also act as amnesics and interfere with learning and memory. Since the Mox did not show any effect of marking off the slits or activity in an open field, it is unlikely to act as a general sedative. However, it was necessary to test the Mox for any adverse effects on learning and memory.

SAW. Moxalactam test for spatial memory

Radial arm maze

The radial arm maze is one of the most commonly used procedures to test rodent space learning and memory. Developed by Olton et al. (1976), it provides the simultaneous choice of several alternative paths for the test subject. Animals should learn which locations provide food (site learning) using visuospatial cues.

Procedures

Experimental trials: Experimental trials consist of three phases (described below). The arms of the labyrinth are listed clockwise from one to seven, with arm number one being the arm furthest from the right side of the labyrinth; All trials last approximately 12 min. When not tested, all hamsters had unlimited access to water. In addition to sunflower seeds in the labyrinth, hamsters are given a daily granule of Agway Prolab 3000 food. Tests within all phases are carried out on successive days.

Phase one: Phase one consists of five 15-minute trials. Before starting 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.

Phase two: Phase two of the experimental trials is identical to phase one, except that the pipes are placed in arms two, four and seven. Arms one, three, five and six remain empty. Phase two consists of four 15-minute trials.

Four and seven primers with sunflower seeds. Phase three differs from phase two in that the labyrinth is rotated 110 ° clockwise in the room.

Behavior coding: An entry was scored in the arm when the four legs of a hamster crossed an arm threshold. A full entry into the arm in one arm is scored when a hamster's snout touches the top of the block at the end of an arm or when its snout passes through the block. These scores were made for arms with bait and without bait. In addition, the number of pipes stored in the abazones by hamsters was scored.

Results

Six male hamsters were tested in the radial arm labyrinth after treatment with 0.9% NaCl or 50 µg / kg of Mox (Fig. 8). Each animal received each treatment and the order of treatments was compensated. The most critical measure in the radial arm labyrinth is the number of pipes discovered after the orientation of the labyrinth is reversed on the final day of the test. Treatment with moxalactam significantly increased the search for pipes (p <0.01) with respect to vehicle treatment.

Summary

These data support the idea that the anxiolytic profile of moxalactam is not accompanied by any learning and memory disorders, as is the case with benzodiazepine anxiolytics. On the contrary, moxalactam enhances spatial memory, it could 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 behavioral disorder in children and senility in geriatric patients.

Space navigation in the water maze

Morris's water maze as well as the radial arm maze was developed to test spatial memory (Morris, 1984). The pool is divided into quadrants normally designated North, South, East and West. The water in the pool becomes opaque with powdered milk. Hidden just below the surface in one of the quadrants is a platform that serves as an escape route for rodents placed in the pool. An animal is placed somewhere in the pool of a variety of different starting points and is timed for latency to find the platform, the time in percentage spent in each quadrant, the distance traveled and the swimming speed. Animals do not have visual or spatial signals in the pool and should be based on additional signs of the maze, that is, objects fixed from the pool that can be observed by the swimming animal. Through a series of tests, a rat develops “site learning” or knowledge about the position of the platform based on the additional signals of the maze. The platform can move every day to a different quadrant by combining spatial memory with working memory. This paradigm implies the extinction of the previous memory and the resolution of a new spatial problem.

Procedures

The water maze consisted of a black plastic circular pool of approximately 150 cm in diameter and 54 cm high filled to a level of 35 cm with water made opaque with powdered milk. The pool was divided into four quadrants with a 10 cm diameter platform submerged 2 cm below the surface in the northwest quadrant. The water was maintained at a temperature of 25 ° C. Around the pool there were several visual cues. Above the pool there was a video camera to follow the movement of the experimental animal. Data collection was fully automated using software developed by HVS Image (Hampton, RU). Before the test, the rats became familiar with the pool and the platform placed in the northwest quadrant. Each day for 4 consecutive days, the animals were placed in the pool at random sites and given two minutes to find the platform. The animals were treated one h before the test with 50 µg / kg of Mox (n = 11) or vehicle (n = 10). After these familiarization tests, the animals were tested for space navigation. The first day of the test began with the platform in the expected northwest quadrant. All behavior was recorded on video during an observation period of two min. After the test the animal was dried and placed back in its home cage. On each subsequent day the platform moved to a new quadrant and the rat started in different positions. The rat was always placed in the pool facing the side wall. The initial positions with respect 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 from the side of the pool and in the left corner from the center facing it.

Results

A bilateral ANOVA showed a significant main effect for treatment (F (1.20) = 6.48, p <0.05) and test days (F (3.63) = 5.76, p <0, 01) (Fig. 19). There was also a significant interaction between treatments and test days (F (3.63) = 4.35, p <01). The Newman-Keuls a posteriori tests showed a significant difference between treatments on day two (p <05), day three (p <01) and day four (p <05) (Fig. 19). On each of these days, animals treated with Mox showed significantly shorter latencies to find the hidden platform than the vehicle treated group. In fact, the animals treated with vehicle showed a significant increase in latency on days 2 (p <, 05) and 3 (p <01) with respect to day 1.

The strategy to find the platform was surprisingly similar for both treatments (Fig. 19, the lower of the two graphs) as judged by the percentage of time the animals spend in each quadrant. For any quadrant on any day there was no significant difference between treatments. There was a significant difference between days for the percentage of time spent in any particular quadrant (for example, North, F (3.63) = 28.80, p <, 0001). The animals spent a significant portion of their time in certain quadrants on certain days. For example, on day 1, both the animals with Mox and the vehicle spent most of their time in the North quadrant with respect to the other quadrants (p <01). This was to be expected since they had knowledge of the location of the platform in this quadrant of the familiarization procedure. Interestingly, the animals with vehicles also spent a significant amount (p <05) of time in the West quadrant on day 1 with respect to the South and East. This was probably because the platform was hidden in the northwestern part of the North quadrant. On day 2, animals with Mox and vehicles spent a significant amount of time in both the North and South quadrants with respect to the East and West. On day 3, animals with Mox do not show a particular trend towards any quadrant, while animals with vehicles still show significant interest in the North quadrant with respect to the South and West. On day 4, the animals with both Mox and vehicle spent most of their time in the correct quadrant (West) with the minimum amount of time in the East quadrant in which the platform hid the day before. This strategy on day 4 shows good spatial, working and procedural memory for both treatments.

The distance covered to reach the platform throughout the days was not significantly different between animals with Mox and with vehicle (Fig. 20). However, animals with Mox showed significantly faster swimming speed than animals with vehicles (F (1.20) = 22.94, p <0001) (Fig. 20). For example, on day 2, both groups moved a distance similar to the platform, except that the animals with Mox covered the distance at almost twice the speed (p <01). Although there was no main effect throughout the days (F (3.63) = 2.27, p <, 09), there was an interaction between swimming speed and days (F (3.63) = 2, 75, p <05) for the treatment with Mox, since this group decreased swimming speed over time.

Navigation with signs in the water maze

Process

The next day after the last day (day 4) of space navigation, the animals were tested for navigation with signals. In these tests, the platform rose above the water level. One h before the test, the animals were treated with Mox or saline vehicle. The same animals that were treated with Mox during space navigation were treated with Mox for navigation with signals. The animals were subjected to a two-minute series of tests with 45 min between trials. In each trial the platform moved to a different quadrant. The study of navigation with signals was identical to that of space navigation, except that the platform was visible and the test was done during five consecutive tests done in a single day. The animals were scored for latency to find the platform, time in percentage spent in each quadrant, path distance and swimming speed for all test periods.

Results

The latency to find the platform was different among the animals treated with Mox and with vehicle (F (1.20) = 24.68, p <0.0001) (Fig. 21). There was also a main effect for days (F (4.84) = 6.53, p <0.0001), but there was no interaction between treatment and days (F (4.84) = 0.99, p <0 ,4). In trials 1, 3 and 4, animals with Mox showed significantly shorter latencies than animals with vehicles.

As in space navigation, the strategy to find the platform was very similar for both treatments (Fig. 21, the lower of the two graphs) as judged by the percentage of time the animals spent in each quadrant. For any quadrant in any trial there was no significant difference between treatments (for example, South, F (1.20) = 1.61, p <0.21). There was a significant difference between trials for percentage of time spent in any particular quadrant (for example, South, F (4.84) = 16.70, p <, 0001). The animals spent a significant portion of their time in certain quadrants in certain trials. For example, in trial 5, both animals with Mox and vehicles spent a significant amount of time in the North quadrant in which the platform was hidden, and in the West quadrant in which the platform had been in the previous test .

Unlike space navigation, the distance traveled during navigation with signals was significantly different between animals with Mox and vehicle (F (1.20) = 44.11 p <0.0001) (Fig. 22). There was also a significant main effect for trials (F (4.84) = 7.90, p <0.0001) and interaction between treatment and traces (F (4.84) = 2.67, p <0.05) . In trial 1 there was no difference in the length of the trajectory between treatments. However, in trials 3 and 4, animals with vehicles moved significantly further to find the platform than animals with Mox. The path length did not change significantly throughout the tests for animals with Mox. Thus, the average travel length in trial 3 for animals with vehicles was significantly greater than for any other trace for this treatment.

Unlike space navigation, there was no significant difference in swimming speed between the two treatments (F (1.20) = 0.67, p <0.42) (Fig. 22). However, there is a main effect throughout the trials (F (4.84) = 17.18, p <0.0001) and an interaction between treatment and trials (F (4.84) = 4.10, p <0.01). In both treatments there is a significant increase in swimming speed with respect to each subsequent trail. For example, from trial 1 to trial 4, animals with Mox and vehicle showed a significant increase in swimming speed (p <0.01).

Summary

Animals treated with moxalactam 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 surprisingly different. During space navigation, animals had to rely on additional labyrinth signals and procedural memory to find the moving platform. It seemed that the animals with Mox and with vehicle showed the same learning and memory since there was no difference in the percentage of time spent in each quadrant during each day of the test. There was no apparent difference in swimming patterns (Figs. 23 and 24). The distance traveled between treatments was not significantly different. Animals with Mox found the platform before, partly because they swam faster. However, navigation with signals presented a different profile. Again, the animals treated with Mox outperformed the vehicles with latency to find the platform. Again, the search strategy as defined by the percentage of time spent in each quadrant was surprisingly similar. However, unlike space navigation, animals treated with Mox showed a much shorter path length. In addition, both treatment groups swam at the same speed.

These data support the idea that the anxiolytic profile of moxalactam is not accompanied by any learning and memory disorders, as is the case with benzodiazepine anxiolytics. On the contrary, moxalactam enhances spatial memory and can 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 behavioral disorder in children and senility in geriatric patients.

VII. Social behavior in nonhuman primates

Experimental procedure

Eight two-year-old male Rhesus male macaques were tested with Mox. The animals were raised with their mothers in a group that was established in a field station. At one year of age they were transferred to individual cages. Each day after this they stayed in pairs for two-three hours. The teenage couples were always the same. This one-year procedure produced adolescent couples or "playmates" who had a well-defined history of social interaction with a dominant state and recognizable subordinate. The demonstration of social behaviors in this provision is very strong due to the limited amount of time the monkeys spent together.

During the experiment, the monkeys were paired in the "game cage" in which they were videotaped for one hour. The study was designed so that behavioral data were obtained for each monkey under treatment with Mox and with vehicle. The treatment was an ABA type administration program: Day 1 - one member of each couple received 0.9% NaCl from vehicle, Day 2 - drug, Day 3 - vehicle. Only one member of a couple was injected on a test day. The other member of a couple was injected a week later under the same ABA program. Moxalactam was injected IM at a dose of 1 mg / kg. Animals were videotaped sixty minutes after injection during an observation period of one h. Animals were scored for more than forty different behaviors (Winslow et al., 1988). Only twenty-eight are listed in Table I. Unreported behaviors, for example, self-bites, vocalizations, stickiness, mounts, escapes, self-grooming, were so infrequent that they were omitted from the analysis. The t test for paired data was performed for each behavior measurement.

Results

The duration of the game fight was significantly reduced (p <0.05) by the Mox treatment with respect to vehicle. This finding was not affected by the social position of the animal, that is, both dominant and subordinate animals showed a decrease in the game fight after the treatment with Mox. Interestingly, several different measures of agonist behavior, for example, composite aggression scores, were grouped together at almost significant levels. It should be noted that these are juvenile Rhesus monkeys and, as such, their expression of social aggression is mainly confined to the game fight. Aggression does not have the same emotional valence as in adults. However, it is believed that the game fight is the youth precedent for adult aggression. Social grooming for adolescent and adult monkeys is the primary measure of affiliate behavior. Although Mox significantly reduced the duration of the game fight, it had no effect on social grooming.

Summary affiliate behavior, remains unchanged. Hence, the finding that Mox can reduce agonist behavior in rodents results in nonhuman primates.

VIII. Moxalactam D and L isomer test

Basis

5 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 normally has biological activity. As the Mox preparation used in these studies is a mixture of the two isomers it was necessary to isolate and test the active isomer.

Procedures

10 The sodium moxalactam salt (FW 564.4) was obtained as a mixed isomer of Sigma Chemical (St Louis MO). D, L-Mox was isolated with HPLC using the procedure explained briefly by Ziemniak et al., 1982. The D, L-Mox was collected in water and fractionated on a C18 column with a 1% MeCN electrophoresis buffer, pH 6.5. The column effluent was monitored at 275 nm with a UV detector. Both isomers came out as individual peaks. The D-Mox had a retention time of 6.7 min, while the L-Mox came out at 8.2 min. Isomers

15 Mox individuals provided are relatively unstable and would rapidly reisomerize during lyophilization making it difficult to have a reasonably pure sample (> 98%). Hence it was necessary to go directly from the HPLC to the animal. Isomer D (approximately 200 µg / ml of HPLC buffer) was diluted to 50 µg / ml of saline and kept on ice until IP injection (50 µg / kg). The L isomer (approximately 150 µg / ml of HPLC buffer) was also diluted to 50 µg / ml of saline and treated similarly.

20 Results

Two groups of eight animals each were tested for offensive aggression after treatment with 50 µg / kg of D- or L-Mox (Fig. 9). The animals were tested 90 min after injection. D-Mox significantly increased latency to bite (p <0.01) and reduced the number of bites (p <0.05). There was no significant difference in the contact time or the marking of slits between the two isomers.

25 Summary

These data identify D-moxalactam as the active isomer that affects offensive-aggressive behavior.

IX. Antibiotic-related antibiotic test for beta-lactam

Basis

Moxalactam is chemically and pharmacologically similar to cephalosporin and penicillin antibiotics. From

In fact, moxalactam is classified as a cephalosporin. The basic structures of all cephalosporins and penicillin are shown below. Each has a beta-lactam ring (A), in turn, cephalosporin has a six-sided dihydrotiazine ring (B) and penicillin has a five-sided thiazolidine ring (B). These basic structures that form the chemical nucleus for these antibiotics naturally occur in fungus. Moxalactam is not found in nature and is characterized by a replacement of the sulfur atom (S) by

35 oxygen in cephalosporin.

Cephalosporin core Penicillin nucleus A -lactam ring A -lactam ring B Dihydrotiazine ring B Dihydrotiazine ring

Cephalosporins and penicillin are bactericidal. Its antibacterial activity is due to an inhibition of the synthesis of peptidoglycans in the bacterial cell wall. Although the exact mechanism of action is not fully understood, these antibiotics bind to several proteolytic enzymes, for example, carboxypeptidases and

endopeptidases, which participate in the synthesis of the reticular structure of the peptidoglycan that strengthens the bacterial cell wall. The interaction between these antibiotics and proteolytic enzymes is reversible. It is believed 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 closely linked with antibiotics, they cannot perform their function and the bacteria lysate as they replicate.

Carboxypeptidases and similar endopeptidases are associated with cell membranes of neurons and glia in the mammalian brain. One of its many functions is to rapidly degrade neuropeptides that act as neurotransmitters. Unlike classical neurotransmitters, for example, dopamine and serotonin, which rely on reuptake mechanisms to stop signal activation, neuropeptides are inactivated by their rapid degradation in the extracellular space. It is believed that these beta-lactam-related antibiotics have psychotropic activity interfering with metabolism (NAALADase activity) in the numerous neuropeptides that alter the brain's neuropeptide environment.

Process

Six animals with equimolar concentrations (90 µM) of moxalactam (Mox), ampicillin (Amp) carbenicillin (Carb), cefoxitin (Cef), amoxicillin (Amox) or saline vehicle were tested. The concentrations were adjusted to match the 50 µg / kg dose used for MOX in previous studies. All solutions were prepared in 0.9% NaCl and IP were administered. The order of the injections was compensated. The animals were tested for offensive aggression 90 min after injection (Fig. 10). There was a significant difference between latency treatments for biting (F (5.30) = 2.83; p <0.45). Both Mox and Amp significantly delayed the latency to bite (p <0.001 and p <0.05, respectively) with respect to vehicle control. There was also a significant difference between treatments in the number of bites (H = 10.6; p <0.05). Both Mox and Amp drugs significantly reduced the number of bites (p <0.05). There was no significant effect of the treatment on contact time or stroke marking (Fig. 11).

Summary

These data indicate that the anti-aggressive effect of the beta-lactam antibiotic Mox may be extended to include beta-lactam ampicillin. Of all the antibiotics tested, Mox has the highest penetrability in the CNS. Patients who were given 2.0 g of Mox IV show levels in the cerebrospinal fluid of drug of approximately 30 µg / ml. The ratio of CSF levels to Mox serum is approximately 15-20%. It is estimated that the serum concentration of Mox in 140 g hamsters who were given an IP injection of 14 µg of drug is 0.1 ng / ml. This would be reflected by a CSF concentration of 15 ng / ml or Mox brain levels of approximately 30 nM. These levels would be almost certain in the range to effectively interact with neuropeptide receptors, most of which have binding affinities in the nanometric range. Interaction with classical neurotransmitters would be less likely because these receptors have Kd in the micro and millimolar range.

Infants with meningitis (conditions that favor the penetrability in the CNS of beta-lactam antibiotics) show a level ratio in CSF with respect to Amp serum of approximately 10%. Cefoxitin, on the other hand, has little penetrability in the CNS even when the meninges are inflamed. Perhaps many of the beta-lactam antibiotics would be effective in suppressing aggressive behavior and are simply limited by their pharmacokinetics and CNS penetrability. To test this idea it was necessary to repeat the beta-lactam antibiotic study using a larger dose of each drug.

X. Beta-lactams at high doses

Six animals with equimolar concentrations (approximately 5 mg / kg; 9 mM) of ampicillin (Amp), carbenicillin (Carb) and cefoxitin (Cef) or saline vehicle were tested. The concentrations were adjusted to match the 5 mg / kg dose used for the dose response study for Mox. All solutions were prepared in 0.9% NaCl and IP were administered. The order of the injections was compensated. The animals were tested for offensive aggression 90 min after injection (Fig. 12). There was a significant difference between latency treatments for biting (F (4.25) = 5.49; p <0.01). Both Amp and Carb significantly delayed the latency to bite (p <0.001) with respect to vehicle control. There was also a significant difference between treatments in the 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 was no significant effect of the treatment on the contact time or the marking of slits (Fig. 13).

Amoxicillin was not included in this study of beta-lactam antibiotics at high doses; instead it was performed in a separate study using a dose of 1 mg / kg (approximately 2 mM). Eight animals were tested for offensive aggression 90 min after IP injection after treatment with Amox or saline vehicle (Fig. 14). Each animal was given each treatment with no less than 48 hours between injections. The treatments were compensated. Aggressive behavior was not significantly altered in animals treated with 1 mg / kg Amox.

Summary

These data indicate that ampicillin and carbenicillin administered in sufficiently high doses can suppress the offensive aggression without altering the contact time or the marking of slits. These data raise the possibility that the psychotropic effect of moxalactam is shared by other beta-lactams and that the mechanisms of biological action may be similar. Bioavailability and penetrability in the CNS may be partly the main component that contributes to differences in biological efficacy. In fact, the most recent evidence has shown that clavulanic acid, a β-lactam compound that has non-clinically significant antibiotic activity, but a clinically important β-lactamase inhibition activity, has a wide variety of psychotropic effects, which includes anti-anxiety, anti-aggression and potentiation of cognition, at ip doses less than 1 µg / kg. Its high oral absorption and good transport properties to the blood brain barrier make it and related β-lactamase inhibitors preferred candidates for use in the methods and pharmaceutical formulations according to the present invention.

It is now believed that the mechanism (s) of action for the psychotropic effects of these beta-lactams is their interaction with neurogenic NAALADase. This is feasible since it is reported that cephalosporins have bactericidal activity in concentrations of only 10 nM. Note, the estimated concentration of Mox in the brain after treatment with 50 µg / kg is approximately 30 nM.

Another possible explanation for the psychotropic activity of beta-lactam antibiotics is the possible blockade of known neurotransmitter receptors or reuptake proteins. To test this second possibility it was necessary to screen Mox for interaction with receptors in a wide range of radioligand binding assays.

XI Moxalactam screening in receptor and transport binding assays

Mox test for interaction with vasopressin V1A and serotonin 5HT1A receptors

Vasopressin and serotonin are both critical neurotransmitters in the control of offensive aggression in male hamsters (Ferris et al., 1998). These two neurotransmitters also participate in the control of human aggression (Coccaro et al., 1998). Vasopressin facilitates aggressive behavior, while serotonin inhibits aggression, partially inhibiting the activity of the vasopressin system. The blockade of vasopressin V1A receptors and the stimulation of serotonin 5HT1A receptors in the anterior hypothalamus blocks offensive aggression (Ferris et al., 1999). As Mox significantly suppresses offensive aggression, it was raised as a hypothesis that it did so by interacting with both one and both of these receptors. To test this idea, Mox was tested in a membrane binding assay for competition for the V1A receptor (Ferris et al., 1994) and in an autoradiography assay for recipients for competition for 5HT1A receptors (Ferris et al., 1999). Moxalactam at a concentration of 1 µM did not significantly displace the binding of I125 HO-LVA (vasopressin ligand) in a hamster liver membrane preparation. Similarly, Mox was ineffective in reducing the specific binding of I125 DPAT (serotonin ligand) to hamster brain tissue sections.

Summary

These data show that moxalactam has no direct interaction with the receptors of vasopressin V1A or serotonin 5HT1A in the hamster. This would suggest that moxalactam is affecting behavior by altering the activity of other neurochemical pathways.

Test of amino acid, adrenergic, serotonergic and dopamine receptors and their transporters

Moxalactam was screened in thirty-six different binding trials by NOVASCREEN, a contract research organization based in Hanover, Mariland. The moxalactam was tested at 100 nM and duplicate samples were run for each of the tests listed on the next page. These trials were chosen because their respective receptor or transporter could play a role in the pathophysiology of mental illness. Moxalactam had no significant effect on any of these binding assays.

Amino acid targets

Benzodiazepine, peripheral

GABA

Agonist site

Benzodiazepine, central

GABA

Glutamate

AMPA site

Kainate site

NMDA, agonist site

NMDA, glycine site [strychnine insensitive]

Wisteria site [strychnine sensitive]

Biogenic aminoadrenergic targets

Adrenergic

α1A α1B α2A (human HT-29 cells) α2B α2c (human recombinants) β1 β2

Biogenic aminoserotonergic targets

Serotonin

5HT1A (human recombinants) 5HT1B 5HT1D 5HT2A (formerly 5HT2) 5HT2C 5HT3 5HT4 5HT6 (recombinants of

rat) 5HT7 (rat recombinants)

Biogenic aminodopaminergic targets

Dopamine D1 D2 (human recombinants) D3 (rat recombinants)

Clozapine

Pickup / Conveyor Targets

Adrenosine

Adrenergic, nopapinephrine Dopamine GABA Glutamate Muscarinics,

Serotonin Hill

Hormone targets

Corticotropin releasing factor

Test for corticotropin-releasing hormone receptor

5 Corticotropin-releasing hormone (CRH or CRF as shown on the next page) is a critical neurohormana in stress regulation. As Mox suppresses impulsivity, aggression and anxiety while enhancing learning and memory, it can act to reduce stress. For this reason, Mox was tested by NOVASCREEN in a CSF binding assay. Moxalactam at a concentration of 100 nM had no effect in this trial.

10 Summary

These data show that moxalactam does not interact directly with many of the receptors and transporters involved in the pathophysiology of aggression and mental illness. This leaves three possible mechanisms of action: 1) interaction with known receptors that were not screened, for example, histamine, acetylcholine and other neuropeptides, 2) interaction with unknown receptor or “orphan receptor,” or 3) interaction

15 with peptidolytic enzymes (for example, NAALADase) in the CNS that alter the chemical environment of the brain.

XII. Review of the mechanism of action

Peptidoglycan precursor peptide test for effects on offensive aggression

Basis

Beta-lactam antibiotics have a stereochemistry that resembles that of acyl-D-alanyl-D-alanine, the substrate

20 natural for bacterial proteolytic enzymes. Supposedly, these structural characteristics allow beta-lactam antibiotics to behave as a competitive substrate that blocks enzymatic activity. To test this hypothesis, an acyl-D-alanyl-D-alanine analogue, the peptidoglycan precursor peptide (Nieto and Perkins 1971; Zeiger and Maurer, 1973) was tested for anti-aggressive effects in the resident / intruder hamster paradigm.

Process

The peptidoglycan precursor peptide, Ala-D--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 approximately 2 mM. The animals were anesthetized with sodium pentobarbital (50 mg / kg), guide cannulas for microinjection directed to the lateral ventricle were implanted and allowed to recover for two days before the test. On the day of the test, the animals (n = 6) were injected with vehicle (2% DMSO in 0.9% NaCl) or PPP in a dose of

30 approximately 1 mg / kg in a volume of 1 µl. Sixty minutes after the injection, the animals were retested for offensive aggression towards a smaller intruder placed in their home cage. Two days later, the animals were tested again, and the order of the treatments was reversed.

Results

The peptidoglycan precursor peptide significantly increased the latency to bite (p <0.05) and reduced the

35 number of bites (p <0.05) during an observation period of 10 min (Fig. 15). There was no significant difference in the contact time or the marking of trips between treatments (Fig. 15).

Peptidoglycan precursor peptide test for olfactory discrimination effects

Six animals received an intracerebroventricular vehicle injection or 1 mg / kg of PPP and were tested for olfactory discrimination by measuring their latency to find hidden sunflower seeds (Fig. 16). Injections were compensated by receiving each animal each treatment. Before the test, the animals fasted for 24 h.

5 Sixty min after the injection, the animals were briefly removed from their home cage while six sunflower seeds were buried under the straw bed in a corner. The animals were placed back in their home cage and scored for latency to find the pipes in an observation period of five min. The latency to find the pipe was significantly (p <0.05) reduced in animals treated with PPP with respect to vehicle.

10 Summary

Direct injection of the peptidoglycan precursor peptide into the hamster brain has the same behavioral results as peripheral Mox injection. Both drugs and both routes of administration significantly reduce aggressive behavior without altering the social interest of motor activity, that is, the contact time and the marking of trips. In addition, the enhancement of olfactory discrimination that seems to be the trial

15 of simpler and more robust behavior to screen beta-lactam antibiotics is similarly affected by the precursor peptide. These findings are proof that beta-lactam antibiotics affect behavior: 1) acting directly on the brain, and 2) resembling the rest of the acyl-D-alanyl-Dalanine peptide.

Although clavulanic acid contains a beta-lactam ring and is structurally similar to penicillins and

20 cephalosporins, has a weak antibacterial activity with no therapeutic value as an antibiotic. However, when administered in combination with some beta-lactam antibiotics such as ticarcillin (Timentin®), 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 inactivate beta-lactam antibiotics (Brown et al., 1976; Reading and

25 Cole 1977).

Clavulanic acid.

Clavulanic acid is commercially available in the United States, but only in fixed combination with other drugs. The commonly prescribed Timentin® is usually administered intravenously at doses ranging from 200-300 mg / kg / day (based on ticarcillin content) that corresponds to a clavulanic acid dose of approximately 7-10 mg / kg / day ( AHFS, 1991). There are no adverse reactions or contraindications reported for clavulanic acid administered in this dose range (Koyu et al., 1986; Yamabe et al., 1987). The data presented below report that clavulanic acid can alter activity in the CNS and behavior at doses ranging from 10 ng to 10 µg / kg, or 1000 to 1.00,000 times lower than those used in

35 antibacterial indications.

Clavulanic acid itself is active and stable orally. The bioavailability is approximately 64 to 75% (Davies et al., 1985; Bolton et al., 1986) with an elimination half-life of just less than two hours. Peak plasma concentrations occur between 45 min and three hours after ingestion (Bolton et al., 1986) with a plasma half-life of more than 2 h (Nakagawa et al., 1994). The volume of distribution is

40 approximately 15 liters, suggesting that clavulanic acid is mainly confined to extracellular fluid (Davies et al., 1985). The CSF / plasma ratio is approximately 0.25, proof that clavulanic acid easily passes the blood brain barrier (Nakagawa et al., 1994).

BEHAVIOR STUDIES WITH CLAVULANIC ACID

I. Response to clavulanic acid dose in the anxiety pipe search model

45 Foundation

Clavulanic acid (CLAV) is structurally similar to beta-lactam antibiotics. A more robust and simple bioassay to screen beta-lactams for activity in the CNS is the anxiety model of searching for pipes by golden hamsters. Briefly, hamsters were deprived of food overnight. The next day they are exposed to the additional stress of being taken out of the 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 hide under the bed of straw in one of the corners. When they return to the home cage, the hamsters routinely stand along the walls for 1-2 min before settling down, locating and eating the pipes. However, animals treated with benzodiazepine chlordiazepoxide anxiolytic find the pipes in less than 10 s. This reduction in the search time for pipes from minutes to seconds also occurs after treatment with moxalactam and other beta-lactam antibiotics.

Experimental protocol

Golden Syrian male hamsters (Mesocricetus auratus) (120-130 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, IN) were individually housed in Plexiglas cages (24 cm x 24 cm x 20 cm), kept in a reverse cycle of light: darkness (14L: 10O; the lights come on at 7:00 p.m.) and food and water were provided at will. A range of CLAV concentrations (saline vehicle, 0.1, 1.0, 10, 100, 1,000 ng / kg) was tested in six groups of hamsters (4-8 / group) (Fig. 25). All tests were performed during the dark phase of the circadian cycle under dim red lighting. Before the test, all animals fasted for 20-24 h. Ninety minutes after intraperitoneal (IP) injection of the drug, the animals were removed from their home cage and placed in a holding cage for 2 min. During his absence, six sunflower seeds were buried under the bed of straw in a corner of his home cage. The animals were placed back in their home cage randomly oriented towards any of the empty corners and their latency was timed to find the pipes in an observation period of five min. Latency times were analyzed with a unilateral ANOVA followed by Scheffe a posteriori tests. The assumption of equal variances was tested (Hartley's Fmax = 2.1 p> 05)

Results

The latency to find sunflower seeds was significantly different between doses (F (5.30) = 10.0; p <0.0001). The CLAV in doses of 10 ng and above significantly reduced (p <0.01) latency times to less than 8.0 s with respect to the saline vehicle with an average latency of 104 s. The dose of 1 ng / kg was not significantly different from that of the vehicle control.

Summary

The data shows that the CLAV administered at a dose of 10 ng / kg of body weight is maximally effective in the pipe search test. The adult male hamsters used in these studies weighed approximately 125 g. Hence, these animals will be administered approximately 1.25 ng of CLAV. The CLAV has a volume of distribution that approximates the volume of extracellular fluid. 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 (the formula weight of the CLAV potassium salt is approximately 240). Since the CSF / plasma ratio is 0.25, the estimated concentration in the brain would be approximately 50 pM.

The pipe search anxiety model seems to have empirical validity (McKinney 1989), that is, drugs such as benzodiazepines used to treat clinical anxiety are effective in the animal model. However, a broader spectrum of anxiolytics and ineffective drugs should be screened to assess the incidence of false negatives and false positives before adopting the search for pipes as an anxiety model. Hence, it was necessary to validate the potential for anxiolytic activity of CLAV in the traditional elevated cross labyrinth.

II. Clavulanic acid test in the elevated cross labyrinth

The elevated cross labyrinth was developed to screen for anxiolytic and angiogenic drugs in the rat (Pellow et al., 1985). The procedure has been validated in terms of behavior, physiologically and pharmacologically. The cross labyrinth consists of two open arms and two closed arms. Rats will naturally make fewer entries in open arms than in closed arms and spend significantly less time in open arms. Confinement in open arms is associated with behavior related to significantly more anxiety and higher levels of stress hormone than confinement in closed arms. Clinically effective anxiolytics, for example, chlordiazepoxide or diazepam, significantly increase the percentage of time spent in open arms and the number of entries in open arms. In contrast, anxiogenic compounds such as yohimbine or amphetamines reduce the entries in the open arms and the time spent in the open arms.

Experimental protocol

Male Wistar rats weighing 250-300 g were housed in a group in a normal 12:12 light-dark cycle with the light on at 08:00 h and food and water were provided at will. The cross labyrinth consisted of two open arms, 50 cm long, 10 cm wide, with 40 cm high walls made of transparent 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 each other to form the cross labyrinth. The labyrinth rose to a height of 50 cm. Eighteen animals were tested in the cross labyrinth 90 min after the IP injection of 1.0 µg / kg of CLAV, 50 or vehicle control in a volume of approximately 0.3 ml. The order of treatments was compensated with at least 48 hours between injections. At the beginning of the experiment, the animal was placed at the end of one of the open arms. During an observation period of five min, the animals were scored for latency to enter the closed arm, time spent in the closed arm and the number of entries in the open arms after the first occupation of the closed arm. The study produced repeated measurement tables. The data between treatments were compared with a bilateral repeated measures ANOVA followed by Bonferroni a posteriori tests.

Results

There was a significant difference between treatments for latency to enter darkness (F (1.18) = 8.53; p <0.01). When treated with CLAV (p <0.05), the animals remained in the position with initial open light longer than when treated with vehicle (Fig. 26). The time spent in the open arm was highly significant between treatments (F (1.18) = 144; p <0.0001) (Fig. 26). The time spent in the open arm increased significantly for CLAV (p <0.01) with respect to the vehicle. Finally, the entries in the open arms were significantly different between treatments (F (1.18) = 44.0 p <0.0001) with CLAV (p <0.01), treatment that shows an increase in movement towards the arms open lights with respect to the vehicle (Fig. 26).

Summary

These data show that the CLAV administered at a dose of 1 µg / kg has anxiolytic activity in the cross labyrinth. These data are encouraging; however, many anxiolytics such as benzodiazepines reduce motor activity. Since animals treated with CLAV needed more time to move from the illuminated 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 delayed movement. To control 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

Immediately after each of the cross-labyrinth tests previously reported in section II, the animals were tested for general motor activity in an "open field." The animals were placed in a large clean Plexiglas cage (48 x 32 x 40 cm) lacking a bed of straw. This open field was delineated in equal quadrants by adhesive tape over the bottom of the cage. The animals were scored for motor activity counting the number of quadrants crossed in 1 min. There were no significant differences between treatment with CLAV and with vehicle in open field activity (Fig. 27).

Summary

There is no evidence in the open field test that the CLAV reduces motor activity. This finding is corroborated in another study of behavior, the marking of slits reported in section VII. Offset marking is a complex stereotyped motor behavior used by hamsters to disseminate pheromones for olfactory communication (Fig. 39). Offset marking is not affected by CLAV treatments. It seems that this beta-lactam has an advantage over the more conventional benzodiazepine anxiolytics because it does not reduce motor activity. However, is the anxiolytic activity of CLAV comparable with that of clinically prescribed benzodiazepines?

IV. Clavulanic acid against chlordiazepoxide in the cross labyrinth

Experimental protocol

Chlordiazepoxide (Librium®) is a commonly prescribed anxiolytic that has been rigorously characterized in preclinical studies. The effective anxiolytic dose in the cross labyrinth is 10-25 mg / kg (Lister 1987; File and Aranko 1988; Shumsky and Lucki 1994). In this dose range, chlordiazepoxide (CDP) is a sedative and reduces motor activity, complicating the interpretation of any behavioral test that requires locomotion (McElroy et al., 1985). However, it was discovered that the animals developed a tolerance to 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) every day for seven days before the start of the experiment. Although 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 ensure a balanced experimental design. In addition, there was a third group of rats (n = 6) that received daily injections of saline vehicle. The study reported in section II tested CLAV at 1 µg / kg in the cross labyrinth. Data from the anxiety pipe search trial shown in section I suggests that CLAV should be effective between doses of 10 ng to 1 µg / kg. For this reason the CLAV was tested at 100 ng / kg in these studies.

Results

There was a significant difference between treatments (F (2.15) = 21.45, p <0.001) for latency to enter darkness. The latency to enter the dark closed arms was significantly higher for animals treated with CLAV and CDP (p <0.01) with respect to vehicle control (Fig. 28A). There was also a significant difference between treatments (F (2.15) = 17.14, p <0.001) for the time spent in the light. The time spent exposed to light in open arms was also significantly longer for animals treated with CLAV and CDP (p <0.01) with respect to the vehicle (Fig. 28A). There was no significant difference between treatments for open arm entries (Fig. 28B).

Summary

These data show that the CLAV and the CDP have similar anxiolytic activity in the elevated cross labyrinth. However, CLAV has greater potency, being more effective at doses 100,000 times lower than CDP. In addition, CLAV does not have the sedative motor depressive activity of conventional benzodiazepine anxiolytics. The anxiolytic effects of CLAV are immediate and do not require the development of tolerance to realize the effectiveness of the behavior. However, a warning point, benzodiazepines have another undesirable side effect for which there is no tolerance development - amnesia (Shumsky and Lucki 1994). For example, diazepam (Valium®) selectively affects short-term memory and attention while avoiding long-term memory (Liebowitz et al., 1987; Kumar et al., 1987). Therefore, it was necessary to test CLAV for any adverse effects on learning and memory.

V. Clavulanic acid and spatial memory in the water maze

Morris's water maze was developed to test spatial memory (Morris, 1984). The pool is divided into quadrants normally designated North, South, East and West. The water in the pool becomes opaque with powdered milk. Hidden just below the surface in one of the quadrants is a platform that serves as an escape route for rodents placed in the pool. An animal is placed somewhere in the pool of a variety of different starting points and is timed for latency to find the platform, the time in percentage spent in each quadrant, the distance traveled and the swimming speed. Animals do not have visual or spatial signals in the pool and should be based on additional signs of the maze, that is, objects fixed from the pool that can be observed by the swimming animal. Through a series of tests, a rat develops “site learning” or knowledge about the position of the platform based on the additional signals of the maze. The platform can move every day to a different quadrant by combining spatial memory with working memory. This paradigm implies the extinction of the previous memory and the resolution of a new spatial problem.

1. Space navigation

Procedures

The water maze consisted of a black plastic circular pool of approximately 150 cm in diameter and 54 cm high filled to a level of 35 cm with water made opaque with powdered milk. The pool was divided into four quadrants with a 10 cm diameter platform submerged 2 cm below the surface in the northwest quadrant. The water was maintained at a temperature of 25 ° C. Around the pool there were several visual cues. Above the pool there was a video camera to follow the movement of the experimental animal. Data collection was fully automated using software developed by HVS Image (Hampton, RU). Before the test, the rats became familiar with the pool and the platform placed in the northwest quadrant. Each day for 4 consecutive days, the animals were placed in the pool at random sites and given two minutes to find the platform. The animals were treated one h before the test with 1.0 µ / kg CLAV (n = 9) or vehicle (n = 9). After these familiarization tests, the animals were tested for space navigation. The first day of the test began with the platform in the expected northwest quadrant. All behavior was recorded on video during an observation period of two min. After the test the animal was dried and placed back in its home cage. On each subsequent day the platform moved to a new quadrant and the rat started in different positions. The rat was always placed in the pool facing the side wall. The initial positions with respect to the platform were different for each of the four trials; however, the platform was always in the same relative position in each quadrant. 20 cm was positioned from the side of the pool and in the left corner from the center facing it. The latency to find the hidden platform, the length of the path, the swimming speed and the times per quadrant among animals treated with CLAV and with vehicle were compared with a bilateral ANOVA of repeated measures followed by Bonferroni a posteriori tests.

Results

There was no main effect for drug treatment (F (1.16) = 4.17, p <0.057), test days (F (3.48) = 0.51, p> 0.5) or interaction between factors (F (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 tendency towards significance.

The strategy to find the platform was similar for both treatments (Figs. 30A and 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 the percentage of time spent in any particular quadrant (for example, CLAV, North Quadrant, F (3.32) = 38.81, p <, 0001). The 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 Northern quadrant with respect to the other quadrants (p <01). This was to be expected since they had knowledge of the location of the platform in this quadrant of the familiarization procedure.

Although the strategy to find the platform as measured by the percentage of time spent in each quadrant was similar between CLAV and vehicle, there was a small but obvious difference. The CLAV treated animals spent more time in the correct quadrant than the vehicle treated animals. This difference is particularly true on day 2 when the animals with CLAV spent more than 50% (p <0.01) of their time in the correct quadrant (South). The animals with vehicles spent less than 40% of their time in the correct quadrant, a time not significantly different from that of the other quadrants. On 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.

There was a significant main effect for the treatment (F (1.16) = 8.40, p> 0.01) on the path length to find the platform. On day 1, the animals treated with CLAV (p <0.05) moved a much shorter distance during the search of the platform than the animals with vehicles (Fig. 31). There was no significant difference between CLAV and vehicle in swimming speed (Fig. 32).

2. Navigation with signals

Process

The next day after the last day (day 4) of space navigation, the animals were tested for navigation with signals. In these tests, the platform rose above the water level. One h before the test animals were treated with CLAV or saline vehicle. The same animals treated with CLAV during space navigation were treated with CLAV for navigation with signals. The animals were subjected to a two-minute series of tests with 45 minutes between trials. In each trial the platform moved to a different quadrant. The study of navigation with signals was identical to that of space navigation, except that the platform was visible and the test was done during five consecutive tests done in a single day. The animals were scored for latency to find the platform, time in percentage spent in each quadrant, path distance and swimming speed for all test periods

Results

There was no main effect for treatments (F (1.16 = 0.553 p> 0.1), trials (F (4.64) = 0.9745, p> 0.1) or interaction between factors (F (4, 64) = 0.7433, p> 0.5) for latency to find the platform during navigation with signals (Fig. 33).

As in space navigation, the strategy to find the platform was very similar for both treatments (Figs. 34A and B) as judged by the percentage of time the animals spent in each quadrant. For any quadrant in any trial there was no significant difference between treatments (for example, Trial 1, North, F (1.16) = 0.099, p> 0.5). There was a significant difference for the percentage of time spent in any particular quadrant for any treatment for most trials, the most notable for CLAV.

The distance traveled to find the platform was not significantly different between the animals treated with CLAV and with vehicle (F (1.16) = 0.23 p> 0.5) (Fig. 35). Although there was no significant main effect for the treatment on swimming speed (F (1.16) = 0.926, p> 0.1), there was a significant trace effect (F (4.64) = 7.87, p <0.001) and interaction between factors (F (4.64) = 2.56, p <0.05). Both treatments, but particularly the CLAV, showed reduced swimming speeds 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 and B.

Summary

Animals treated with clavulanic acid do not show any loss in learning and memory when tested for space navigation and with signs in the Morris water maze. In fact, in the distance traveled to the hidden platform and the percentage of time spent in the correct quadrant for both space navigation and signals, the CLAV treated animals showed better performance than the vehicle. These data show that CLAV's anxiolytic profile is not accompanied by any learning and memory disorders, as is the case with benzodiazepine anxiolytics.

STUDIES ON THE MECHANISMS OF ACTION

SAW. Clavulanic acid and stress response

Basis

The ability of the CLAV to reduce anxiety in stressful situations, that is, the deprivation of food and the novel environment in the pipeline search trial, and exposure to light and novel surroundings in the raised cross labyrinth, without altering the 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 expanded if we had a clearer understanding of its mechanism of action. For example, could CLAV be altering anxiety by suppressing the response to natural stress? Commonly prescribed benzodiazepine anxiolytics block both normal circadian release and stress-mediated release of the hormone cortisol (Gram and Christensen, 1986; Petraglia et al., 1986; Hommer et al., 1986).

Experimental protocol

The simple procedure of placing an adult male hamster in a novel environment for 5 min produces a predictable significant increase in cortisol blood levels (Weinberg and Wong 1986). This novel test was used to assess the effects of CLAV on stress-induced release of cortisol. Two groups of male hamsters with both CLAV (10 µg / kg, n = 6) and saline vehicle (n = 4) were treated IP. A third group (n = 4) received no treatment or stress due to isolation and served as a control for baseline cortisol levels. Sixty min after treatment, the animals were removed from their home cage and placed in a novel cage for 5 min. The animals were then sacrificed by decapitation and trunk blood was collected for cortisol radioimmunoassay. All animals were tested under light conditions: dark reverse four hours in the dark cycle. Data were compared with a unilateral ANOVA followed by Fisher PLSD tests a posteriori.

Results

There was a significant difference in stress release of cortisol between treatments (F (2.11) = 10.03 p <0.01). The vehicle (p <0.05) and the CLAV (p <0.01) showed more than twice the level of cortisol blood with respect to the untreated untreated control (Fig. 37).

Summary

The data shows that the CLAV beta-lactam anxiolytic has no apparent effect on cortisol release in response to mild stress from exposure to a novel environment. This detail, combined with the absence of motor depression and cognitive impairment makes CLAV unique among the anxiolytics and suggests a novel highly specific mechanism of action. At first glance one might think that it would be advantageous to suppress the stress response. In fact, hypercortisolism participates in the pathophysiology of depression (Sacher et al., 1973). Chronic psychosocial stress that leads to dysfunctional hyperactive adrenal glands can be life-threatening. However, a sensitive hypothalamus-pituitary-adrenal axis is critical for normal physiology and behavior. Stressors that would normally help animals adapt to the environment can be deadly without proper cortisol release.

VII. Territorial or offensive aggression

Basis

Continuing to study the activity in the CLAV SNC in more complex behavior models can help clarify its mechanism (s) of action. For example, antagonistic social interactions between animals require risk assessment, communicative and agonist behaviors to settle disputes over territory, couples, food, etc. The neurotransmitters serotonin and vasopressin are fundamental in the organization of the CNS and the expression of these behaviors in animals and humans (Ferris et al., 1997; Coccaro et al., 1998; Ferris 2000). To this end, the CLAV was tested for effects on territorial or offensive aggression, that is, defense of the home burrow against intruders.

Agonist behavior can be classified as both offensive and defensive aggression (Blanchard and Blanchard, 1977; Adams, 19798; Albert and Walsh, 1984). Offensive aggression is characterized by an aggressor who initiates an attack on an opponent, while defensive aggression lacks active focus. Both types of aggression have their own unique behavioral systems. The stimuli that provoke the offensive and defense attack are different, since they are the sequences of behaviors that accompany each agonist response. While many of the empirical data that support the idea 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 golden male 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 a strange male hamster in the home cage of another male hamster causes a well-defined sequence of resident agonist behaviors that includes offensive aggression.

Experimental protocol

Hamsters are nocturnal and as such all behavioral tests were performed during the four

First 5 hours of the dark phase under dim red lighting. The resident was scored for offensive aggression, for example, latency to bite the intruder, the total number of bites, total contact time with the intruder and the marking of slits during a 10-minute trial period (Ferris and Potegal, 1988) . Ijada marking is an olfactory communication form in which a hamster arches its back and rubs the glands of the pheromone-producing ijada against objects in the environment (Johnson, 1986). The frequency of tagging is greatly enhanced during aggressive encounters and is particularly strong in dominant animals that start and win fights (Ferris et al., 1987).

Five golden male hamsters (130-140 g) were given IP injections of CLAV (200 µg / kg) and saline vehicle in a volume of approximately 0.2 ml. In pilot studies it was discovered that the CLAV administered IP at 1.0 µg / kg had no effect on aggressive behavior. Therefore, it was necessary to test CLAV

15 at a higher concentration, but in a dose range that was still acceptable for pharmaceutical studies in aggressive behavior. The vehicle and CLAV treatments were compensated and randomized so that the five animals received each separate treatment for at least 48 h. Animals were tested 90 min after treatment for an observation period of 10 min. Latencies and contact time were analyzed with a bilateral ANOVA. The non-parametric data, that is, the number of bites and the marks of the slits were analyzed by the Wilcoxon test with sign of matched pairs.

Results

Although there was no significant main effect for drug treatment (F (1.3) = 7.40, P <0.07) for latency to bite the intruder, there was a tendency towards significance (Fig. 38). There was no significant main effect for drug treatment (F (1.3) = 2.85, p> 0.1) at the time of contact with the intruder (Fig. 38). There was a difference

25 significant between drug treatment (T = 3.0, p <0.05, N = 8) and the number of bites in the intruder. The CLAV treatment reduced the median number of bites to six with respect 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 departure marking behavior (Fig. 39).

Summary

Clavulanic acid has modest anti-aggressive or serenic-like properties. Serénicos are drugs used to treat impulsivity and violence (Olivier and Mos, 1991). Serénicos should suppress offensive aggression without interfering with social, appetite and cognitive behaviors. The social interest in an intruder, that is, the contact time, is not altered by CLAV. The 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.,

35 1994). The powerful anxiolytic activity of CLAV excludes this possibility.

VIII. Interactions with glutamyl carboxypeptidase

CLAV has a very high binding affinity for beta-lactamases. It is hypothesized the presence of mammalian homologies with these bacterial enzymes and that these homologous proteins participate in the regulation of neurotransmitter levels in the CNS. The E. coli beta-lactamase TEM has been sequenced cloned and crystallized to determine the active site motifs. The four putative binding sites on betalactamase that could accommodate CLAV are designated active site I, II, III and IV. These active sites, sequence localization and amino acid sequences (AA) are as follows:

Active site I:

35 AA in the 3 'direction from the N: STTK end

45 Active site II:

57 AA at address 3 'from the STTK reason: SGC, SGN or SAN

Active site III:

III AA in the 3 'direction from the SGC reason: KTG

Active site IV:

41 AA at address 3 'from the SGC reason: ENKD

Screening for amino acid sequence homologies between these beta-lactamase and mammalian enzyme binding sites. Revaax scientists identified an enzyme system in the brain to which CLAV would possibly bind in a similar way to beta-lactamase. The enzyme glutamyl carboxypeptidase (N-acetyl, alpha-linked, acid dipeptidase) or NAALADase (Pangalos et al., 1999) is responsible for the regulation of 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 overlapping of putative active beta-lactamase sites and the sequences conserved in human and rat NAALADase, the hypothesis was raised that CLAV affected behavior by inhibiting NAALADase activity. The similarity of overlap sequences between beta-lactamase and NAALADase is shown below:

Active site I:

Beta-lactamase: 35 AA in the 3 'direction from the N: STTK end

NAALADasa: 38 AA in the 3 'direction from the N: STQK end

Active site II:

Beta-lactamase: 57 AA in the 3 'direction from the STTK motif: SGC, SGN or SAN

NAALADasa: 59 AA in the 3 'direction from the STQK motif: SFG

Active site III:

Beta-lactamase: 111 AA in the 3 'direction from the SGC motif: KTG

NAALADasa: 110 AA in the 3 'direction from the SFG motif: KLG

Active site IV:

Beta-lactamase: 41 AA in the 3 'direction from the SGC motif: ENKD

NAALADasa: 41 AA in the 3 'direction from the reason SFG: ERGV

Clavulanic acid inhibits gram-negative beta-lactamase enzymes in the range of 15-34 nM. The CLAV is effective at a dose of 10 ng / kg in the pipe anxiety search model (pg 3). If NAALADase were the human homologue for beta-lactamase, then it would be predicted that CLAV would be a high affinity substrate.

IX. Search for pipes after blocking NAALADasa activity

Background and experimental procedure

The hypothesis was raised that the CLAV would work as an anxiolytic in the pipeline search trial blocking the activity of NAALADase in the brain. If this idea were true, then it would be predicted that drugs known to block NAALADase would also enhance the search for pipes. For this purpose, the 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 search anxiety model of pipes. The study was similar to that explained briefly 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 transported by the cerebrospinal fluid through the brain through the ventricular system. Beta-NAAG (FW 304) was administered in a dose of 3 ng in a volume of 1 µl of ICV saline. The average adult hamster brain weighs approximately 1.2 g of which 22% is extracellular fluid. The estimated beta-NAAG concentration was 11 ng / ml or 36 nM.

Two groups of six animals each fasted overnight as previously described and tested the next day. One group was treated with beta-NAAG and the other with saline vehicle and one hour later the latency was timed to search for hidden sunflower seeds. For statistical comparisons, a Student's t-test was used for independent data.

Results

The difference in latency to find the pipes was significantly (p <0.001) different between treatments (Fig. 40). In fact, none of the six microinjected animals with saline vehicle found the pipes in the five-minute observation period. However, three days later, when these same animals were microinjected with beta-NAAG (3 ng / µl) and tested for pipes, they showed a mean latency of 21.8 ± 9.7 s.

Summary

The data shows that beta-NAAG, a specific NAALADase inhibitor, can dramatically reduce latency to search for hidden sunflower seeds, a biological activity shared by CLAV. Since beta-NAAG was active in the pipe search anxiety model, 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, the animals were tested for offensive aggression in the resident intruder paradigm as described in section VII. As reported before, when administered in high concentrations, the CLAV only has a modest effect on the offensive. Although CLAV can enhance the search for pipes at a dose of 10 ng / kg, it only has a modest effect on offensive aggression even with doses up to 200 µg / kg. If beta-NAAG and CLAV share a common mechanism, then beta-NAAG should have little or no effect on aggression.

X Effect of the NAALADase blockade on offensive aggression

Experimental procedure

The animals tested in this study were those used in section IX. After the pipe search test, animals treated with beta-NAAG (n = 6) and the saline vehicle (n = 6) continued in their home cage and were presented with a smaller male intruder. The resident was scored for latency to bite, bites, contact time and marking of slits during an observation period of 10 min. The latency to bite and the contact time between treatments were compared with Student's t-tests. The non-parametric measures of bites and marks of the slits for beta-NAAG in front of the vehicle were compared with Mann-Whitney.

Results

There were no significant differences between animals treated with beta-NAAG and with vehicle for any measure of offensive aggression (Figs. 41 and 42).

Summary

Blocking NAALADase activity with beta-NAAG does not alter offensive aggression as proven in the resident intruder paradigm. This finding is consistent with the idea that CLAV and beta-NAAG share a common mechanism - blocking NAALADase activity.

SEQUENCE LIST

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Claims (19)

1. A β-lactam compound selected from: i) clavulanic acid or a pharmaceutically acceptable salt thereof; or ii) moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof, for use in the treatment of a patient in need of relief from a selected disorder of anxiety, depression, dementia, Alzheimer's disease or Parkinson's disease.

2.

 The β-lactam compound for use as in claim 1, wherein the disorder is anxiety.

3.

 The β-lactam compound for use as in claim 2, wherein the anxiety is anxiety disorder, panic disorder with agoraphobia, panic disorder without agoraphobia, agoraphobia without a history of panic disorders, social phobia, simple phobia , organic anxiety disorder, psychoactive substance anxiety disorder, separation anxiety disorder, childhood or adolescent avoidance disorder, or hyperansitivity disorder.

Four.

 The compound for use as in claim 1, wherein the disorder is depression.

5.

 The compound for use as in claim 4, wherein the depression is major depression or psychotic depression.

6.

 The compound for use as in claim 1, wherein the disorder is dementia.

7.

 The compound for use as in claim 6, wherein the dementia is presenile dementia, senile dementia, multi-infarct dementia, alcohol dementia, psychoactive substance dementia or primary degenerative dementia of the Alzheimer type.

8.

 The compound for use as in claim 1, wherein the disorder is Alzheimer's disease.

9.

 The compound for use as in claim 1, wherein the disorder is Parkinson's disease.

10.

 The compound for use as in any one of claims 1 to 9, wherein the β-lactam compound is moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof.

eleven.

 The compound for use as in claim 10, wherein less than an antibiotically effective amount of moxalactam is administered.

12.

 The compound for use as in any one of claims 1 to 11, wherein the β-lactam compound is clavulanic acid or a pharmaceutically acceptable salt thereof.

13.

 A pharmaceutical formulation for use to treat anxiety, depression, dementia, Alzheimer's disease

or Parkinson's disease in a human patient in need of relief thereof, in which the formulation comprises a β-lactam compound selected from: i) clavulanic acid or a pharmaceutically acceptable salt thereof; or ii) moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof.

14.

 The pharmaceutical formulation for use as in claim 13, wherein the pharmaceutical formulation is an oral pharmaceutical form.

fifteen.

 The pharmaceutical formulation for use as in claim 13 or 14, wherein the β-lactam compound is moxalactam or a pharmaceutically acceptable salt thereof or an active ester form thereof.

16.

 The pharmaceutical formulation for use as in claim 13, wherein the β-lactam compound lacks substantially antibiotic activity.

17.

 The pharmaceutical formulation for use as in claim 13 or 14, wherein the β-lactam compound is clavulanic acid or a pharmaceutically acceptable salt thereof.

18.

 The pharmaceutical formulation for use as in any one of claims 13 to 17, wherein the pharmaceutical formulation is a prolonged release pharmaceutical form.

19.

 The pharmaceutical formulation for use as in claim 13, wherein the pharmaceutical formulation is a parenteral pharmaceutical form.