WO2007087637A2

WO2007087637A2 - Compositions and methods using matrix metalloproteinase (mmp) inhibitors for treating cognitive impairment characterized by persistent or sustained mmp expression and/or activity

Info

Publication number: WO2007087637A2
Application number: PCT/US2007/061167
Authority: WO
Inventors: Joseph Harding; John Wright; Peter Meighan; Stala Meighan
Original assignee: Washington State University Research Foundation
Priority date: 2006-01-26
Filing date: 2007-01-26
Publication date: 2007-08-02
Also published as: WO2007087637A3

Abstract

Particular aspects provide methods for treating cognitive impairment, comprising administration of a therapeutically effective amount of at least one matrix metalloproteinase (MMP) inhibitor sufficient to provide for at least one of precluding, alleviating, reversing, or inhibiting cognitive impairment characterized by sustained elevated expression and/or activity of at least one MMP. Persistent elevations in the expression of MMPs in the hippocampus of aged individuals have a detrimental effect on hippocampal synaptic plasticity, and are an underlying cause of cognitive deficits associated with ageing, and particular aspects therefore provide novel compositions and methods comprising use of MMP (e.g., MMP3 and/or MMP9, etc.) inhibitors to treat age-related cognitive impairment (e.g., cognitive decline). Additional embodiments comprise inhibition of MMP-3 and at least one other MMP (e.g., MMP-9, etc). Exemplary MMP inhibitors comprise a zinc-binding hydroxamate moiety (or a non-hydroxamate zinc-binding moiety), and a peptide or peptidomimetic backbone capable of binding at least one MMP.

Description

COMPOSITIONS AND METHODS USING MATRIX METALLOPROTEINASE (MMP)

INHIBITORS FOR TREATING COGNITIVE IMPAIRMENT CHARACTERIZED BY

PERSISTENT OR SUSTAINED MMP EXPRESSION AND/OR ACTIVITY

FIELD OF THE INVENTION

Aspects of the present invention relate generally to cognitive impairment, matrix metalloproteinases (MMPs) and MMP inhibitors, and more particularly to novel compositions and methods using MMP inhibitors to treat cognitive impairment, where, as disclosed in inventive aspects herein relating to cognitive decline in aged individuals for example, said cognitive impairment is characterized by persistent or sustained elevated expression and/or activity of at least one MMP, and where said methods comprise inhibition of at least one such characteristic MMP (e.g., MMP-3 and/or MMP- 9, etc.).

BACKGROUND

Dementia and Aging. Aging is often accompanied by a decline in neuronal function and plasticity. This decline may be of sufficient magnitude to induce a progressive deterioration in cognitive processing that is evidenced as diminished capacity to learn and consolidate memory. Such changes can result in senescence- associated cognitive impairment that, in extreme cases, leads to dementia..

Dementia presently affects approximately 10 million people in the U.S. Alzheimer's disease is one of the several categories of dementia (Frontotemporal, Diffuse Lewy Body, Cortcobasal, Parkinson Disease-related, Vascular), and makes up about 50% of the cases. The number of Alzheimer's disease patients is estimated to be 4.2 to 5.8 million in the United States, with a prediction of about 16 million by 2050 as the population ages.

A number of different drugs are currently approved in the U.S. for treatment of dementias including Alzheimer's disease. All have varying numbers and degrees of side-effects including dizziness, nausea, confusion, and headache, as well as cataracts and urinary incontinence. These compounds are approved for use in early to middle stage Alzheimer's patients but only marginal improvement in cognitive processing has been achieved in the majority of patients.

Therefore, there is a pronounced need in the art for novel compositions, methods and approaches to treat indications such as decline in neuronal function and plasticity, progressive deterioration in cognitive processing, cognitive impairment, dementia, Alzheimer's disease, etc.

SUMMARY OF THE INVENTION

According to particular surprising aspects of the present invention, persistent elevations in the expression of matrix metalloproteinases (MMPs) in the hippocampus of aged individuals have a detrimental effect on hippocampal synaptic plasticity, and are an underlying cause of cognitive deficits associated with aging.

Particular aspects of the present invention therefore provide novel methods and compositions having substantial utility for treatment of cognitive impairment (e.g., dementia, cognitive decline in aged individuals, Alzheimer's disease, etc.). In particular aspects, administration of inhibitors of matrix metalloproteinases (MMPs) is herein disclosed to have substantial utility to reverse cognitive decline in aged individuals.

The novel methods have utility for treating a disease or disorder of the peripheral or central nervous system characterized by persistent or sustained expression and/or activity of at least one MMP, including but not limited to Alzheimer's disease, stroke/cerebral ischemia, head trauma, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, migraine, cerebral amyloid angiopathy, AIDS, age-related cognitive decline; mild cognitive impairment and prion diseases in a mammal, which comprises administering to said mammal a therapeutically effective amount of a MMP inhibitor. Preferably, inhibitors of

MMP-3 and/or MMP-9 are used.

Particular embodiments provide a method for treating cognitive impairment, comprising administration to a subject in need thereof a therapeutically effective amount of at least one matrix metalloproteinase (MMP) inhibitor sufficient to provide for at least one of precluding, alleviating, reversing, or inhibiting cognitive impairment characterized by persistent or sustained elevated expression and/or activity of at least one MMP.

Particular aspects comprise administration of at least one broad spectrum MMP inhibitor for treatment of age-related cognitive impairment (e.g., age-related cognitive decline). Additional aspects comprise administration of specific or substantially specific MMP inhibitors for such treatment. In particular aspects, inhibitors of MMP3 and/or MMP9 are herein disclosed to have substantial utility to treat (e.g., preclude, alleviate, reverse or inhibit, etc.) cognitive decline in aged individuals. Particular aspects comprise administration of an MMP-3 inhibitor for such treatment.

Additional embodiments provide methods comprising inhibition of MMP-3 and at least one other MMP that is expressed in the brain (e.g., MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11 , MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20, etc.). Particular embodiments comprise administering at least one inhibitor to affect inhibition of MMP-3 and MMP-9, or at least MMP-3 and MMP-9.

In particular aspects, the at least one metalloproteinase (MMP) inhibitor comprises a hydroxymate moiety. In certain aspects, the at least one metalloproteinase (MMP) inhibitor comprises a zinc-binding hydroxamate moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase. In additional embodiments, the at least one metalloproteinase (MMP) inhibitor comprises a non-hydroxamate zinc-binding moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase.

Yet further embodiments provide combination therapies or treatments, wherein the at least one matrix metalloproteinase (MMP) inhibitor is used in conjunction with at least one additional therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-D show effects of age on fEPSP characteristics and LTP. Acute hippocampal slices were generated from 3-month old (young) and 22-month old (aged) Sprague-Dawley rats. FIGURE 1A shows time-courses of theta burst stimulated LTP from slices of both young (open circle) and aged (grey circle) rats; tetanus indicated by arrowhead. Data expressed as mean +/- S. E. M.

FIGURE 1B shows a comparison of the magnitude of fEPSP amplitude, for young (open bars) and aged (grey bars) during LTP induction and early-maintenance phases. Induction was calculated as the average amplitude from t=4.5 to t=5.5 min following tetanization; early maintenance was calculated as the average amplitude from t=27.5 to t=28.5 following tetanization. Statistical analysis by two factor ANOVA (age x phase) indicates an independent effect of age on fEPSP amplitude (p<.01).

FIGURE 1C shows a dynamic shift of peak-to-peak latency following tetanization during LTP induction (t= 3-6min post tetanization) and early maintenance (t= 25-30min post tetanization). Data expressed as mean +/- S.D. Due to violation of equal variance assumption, data analyzed with Holmes corrected Aspin-Welch tests (young-adults versus aged-adults: ***p<.0001). Sample waveforms provided from slices of both young (top waveforms) and aged (bottom waveforms), for both baseline (solid lines) and potentiated (hashed lines) conditions.

FIGURE 1 D shows baseline latency from peak-presynaptic fiber volley to peak fEPSP (peak-to-peak latency) and baseline amplitudes for slices from both aged and young rats (data expressed as percent young +/- SEM). Due to violation of equal variance assumption for peak-to-peak latency, data were analyzed with Aspin-Welch test (^**^*p<.0001). Baseline amplitudes were analyzed with two sample t-test (p>.05). Sample waveforms provided from both young (solid line) and aged (hashed line).

FIGURES 2A-D show aged rats exhibited greater basal hippocampal MMP-3 levels and MMP-3 / TIMP-2 complex formation than younger rats. FIGURE 2A shows Western blot analysis of hippocampal MMP-3 protein from 3- month old Sprague Dawley rats. Tissue was collected either δminutes, 4 hours or 24 hours subsequent to first day training in Morris Watermaze.

FIGURE 2B shows Western blot analysis of hippocampal MMP-3 levels from 6 month old and 24 month old Sprague-Dawley rats.

FIGURE 2C shows the results for tissues that were also examined for MMP-3 / TIMP complex formation. Identity of MMP-3 / TIMP-2 complex was verified by immunoprecipitating with anti MMP-3 and subsequently probing with anti TIMP-2 (FIGURE 2D). FIGURES 3A, B show that MMP inhibition enhanced hippocampal LTP in slices from aged rats. Acute hippocampal slices were generated from 22month Sprague- Dawley rats. Following initial recovery from dissection, slices were incubated in the presence or absence of FN-439 for four hours. Subsequent to a one hour washout of FN-439 in the recording chamber, LTP was induced by TBS. FIGURE 3A shows a time-course of fEPSP amplitudes for FN-439 treated

(closed circle) or control (open circle). Data expressed as mean values +/- S. E. M.

FIGURE 3B shows area under curve as was calculated from randomly selected fEPSP waveforms, for both FN-439 treated (filled bars) and control slices (open bars), between 3-5min post tetanus, 13-16 min post tetanus, and 25-30 min post tetanus. Data expressed as mean values +/- S. E. M. Analysis by two factor ANOVA (LTP phase x treatment) revealed an independent effect of FN-439 on area under curve (p<.001) n=16 waveforms per group.

FIGURES 4A, B show that MMP inhibition promoted young-like fEPSP characteristics in slices from aged rats. Slices generated from aged rats were pretreated with FN-439 were assessed for alterations to fEPSP characteristics.

FIGURE 4A shows peak-to-peak latency (open circles) and mean response amplitude (filled circles) during baseline transmission for FN-439 treated and untreated conditions. Data expressed as % change from control (untreated) conditions (mean +/- S.E.M.). Each pair of data analyzed with two-sample T-Test. MMP inhibitor treated slices experienced a significant increase in baseline peak-to-peak latency (***p<.0001); this difference was not reflected in baseline response amplitudes (p>.05). Provided are sample waveforms for both MMPi treated (hashed line) and control (solid line) slices generated from an individual aged animal. Arrowhead denotes presynaptic fiber volley (1^st peak) for both treated and control slices, solid arrow approximates peak from untreated slice, hashed arrow approximates peak from treated slice.

FIGURE 4B shows the dynamic shift of peak-to-peak latency during induction (3- 5 min post tetanization) and early maintenance (25-30 post tetanization) phases. Data expressed as mean +/- S. D. Analysis with two-factor ANOVA (treatment x time) indicates an independent effect of FN-439 on latency shift (p<.0001). Included are sample waveforms for both control (top) and MMPi treated (bottom) conditions during baseline (solid line) and early maintenance (hashed line) phases.

FIGURES 5A-E show that MMP inhibition failed to enhance LTP or alter fEPSP temporal-characteristic in slices from young rats. MMPi pre-treated slices generated from young rats were assessed for LTP.

FIGURE 5A shows the LTP time-course of amplitudes for MMPi treated (filled circle) and untreated (open circle) conditions. Data expressed as % change from baseline (mean +/- S.E.M.).

FIGURE 5B shows the pooled amplitudes for both control (open bars) and MMPi treated (closed bars) during induction (3-5 min post tetanus) and early maintenance (25-30 min post tetanus). Data expressed as % change from baseline (mean +/- S.E.M.).

FIGURE 5C shows the area under curve during induction and early maintenance phases. Data expressed as % change from baseline (mean +/- S.E.M.). FIGURE 5D shows the baseline peak-to-peak latency for MMPi treated and control slices generated from young rats. Data expressed as % change from control (untreated) conditions. MMP inhibition failed to alter baseline peak-to-peak latency (two sample T-test; p>.05). FIGURE 5E shows the Dynamic latency shifts for both control and MMPi treated conditions in slices generated from young rats during induction and early maintenance. Data expressed as mean +/- S.D. MMP inhibition failed to impact tetanus induced latency shift (two factor ANOVA (treatment x phase); p>.05). FIGURES 6A-C show that exogenous MMP-3 application impaired hippocampal

LTP maintenance. Acute hippocampal slices were generated from 3-month old Sprague-Dawley rats. Thirty minutes prior to tetanization, slices were exposed to active MMP-3 (which was present during duration of time-course). LTP was induced by TBS (indicated by arrowhead). FIGURE 6A shows a time-course of fEPSP amplitudes for MMP-3 treated (closed circle) or control (open circle).

FIGURE 6B shows the area under curve as was calculated from randomly selected fEPSP waveforms, for both MMP-3 treated and control slices, between 3-5min post tetanus (induction) and 25-30 min post tetanus (maintenance). Data expressed as mean +/- S.E.M.

FIGURE 6C shows the time-to-peak and amplitude during baseline stimulation for both control and MMP-3 treated slices. Exposure to active MMP-3 significantly decreased baseline peak-to-peak latency (two sample T-test; *p<.05).

FIGURES 7A, B show the demonstrated effects of H₂O₂ exposure on MMP-3 activity regulation in acute hippocampal slices. Twelve 200 micron Hippocampal slices from 6 month old rats were randomized and pre-incubated in ACSF for 2 hours prior to treatment. Six slices were treated with .6mM H₂O₂ in ACSF for 6 hours and compared to six slices from the same hippocampus, incubated in ACSF + vehicle for 6 hours.

FIGURE 7A shows that H₂O₂-treated slices (lane 2) observed an increased MMP-3 / TIMP-2 complex formation as compared to control slices (lane 1). FIGURE 7B shows data expressed as mean values (+/- SEM) n = 3. FIGURE 8 shows that hippocampal cortactin levels were demonstrated to be regulated by MMP activity. Training in the Morris water maze for one day significantly reduced hippocampal cortactin levels as compared to home cage controls (n=8, *p=0.035). MMP inhibition with FN-439 reversed water maze associated cortactin down-regulation and increased cortactin levels well above baseline. (n=6 per group). (*p<0.05, ***p<0.001 , significantly different than vehicle control) Accompanying blot images demonstrate the typical cortactin doublet immunoreactivity at approximately 80 and 85 kDa.'

FIGURE 9 shows that cortactin levels were demonstrated to be depressed in aged hippocampal tissue. Hippocampal tissue from 3, 12 and 24 month old rats was assessed for cortactin levels by Western blotting. 24 month old animals have severely attenuated hippocampal cortactin levels during a time in which they have chronic elevations in MMPs. This corresponds with applicants' data (FIGURE 8) demonstrating an inverse relationship between MMP activity and cortactin.

FIGURE 10 shows an that active MMP-3 protein inversely correlates with hippocampal mass. Hippocampi from 24 month old Sprague-Dawley rats were dissected and weighed prior to assessment of MMP levels by Western blot analysis. Mass and MMP-3 levels were subjected to linear regressϊojn analysis. Rats demonstrated an inverse relationship between hippocampal MMP-3 levels and hippocampal mass (p=.011).

FIGURES 11A₁B show effects of MMP inhibition on water maze latencies for aged-adult rats. Fisher 344 (F344) rats were subjected to five days of Morris water maze training (4 maze trials per day). MMP inhibitor treated rats were given a daily IP injection of FN-4392-hours prior to water maze training.

FIGURE 11A shows the distribution of mean latencies represented as box plots; the box represents the inter-quartile range (IQR; 25^th to 75^th percentiles), the median value, and the adjacent values [defined as (1.5) x (IQR)J. Green and red filled circles represent moderate outliers [(1.5-3.0) x (IQR)] and severe outliers [(> 3.0) x (IQR)] respectively. C1-C5 corresponds to control animal latencies on days i-5 and FN1-FN5 corresponds to MMP inhibitor treated animal latency for days 1-5 respectively.

FIGURE 11B shows the mean values (+/- SEM) as expressed for both control (open circles) and MMPi treated (filled circles). Data fit with exponential decay functions for both treated and control groups. Analysis with two-factor ANOVA (treatment vs. day) reveals an independent treatment effect on daily mean latencies (P=.0063).

DETAILED DESCRIPTION OF THE INVENTION

According to particular surprising aspects of the present invention, persistent elevations in the expression of matrix metalloproteinases (MMPs) in the hippocampus of aged individuals have a detrimental effect on hippocampal synaptic plasticity, and are an underlying cause of cognitive deficits associated with aging. Aspects of the present invention, therefore, relate to a novel methods of treating a disease or disorder of the peripheral or central nervous system, including but not limited to Alzheimer's disease, stroke/cerebral ischemia, head trauma, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, migraine, cerebral amyloid angiopathy, AIDS, age-related cognitive decline; mild cognitive impairment and prion diseases in a mammal, where said methods comprise administering to a subject in need thereof (e.g., a mammalian subject) a therapeutically effective amount of at least one MMP inhibitor. In particular aspects, inhibitors of MMP-3 and/or MMP-9 are used.

Particular aspects of the present invention therefore provide novel methods and compositions having substantial utility for treatment of cognitive impairment, including but not limited to age-related cognitive decline. In particular aspects, inhibitors of matrix metalloproteinases (MMPs) are herein disclosed to have substantial utility to reverse cognitive decline in aged individuals.

In preferred aspects, inhibitors of MMP3 and/or MMP9 are herein disclosed to have substantial utility to treat or reverse cognitive decline in aged individuals. Administration of specific or substantially specific MMP-3 inhibitors for treatment of age- related cognitive impairment (e.g., age-related cognitive decline) is a particularly preferred embodiment of the present invention. Additional embodiments provide methods comprising inhibition of MMP-3 and at least one other MMP (e.g., MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20, etc.).

Overview:

Extracellular matrix (ECM) and Matrix Metalloproteinases (MMPs). Extracellular matrix (ECM) molecules are believed to play an important role in the process of neural plasticity that mediates learning and memory. For example, the ECM is a dynamic network that provides support for neurons and glia and is also involved in a wide range of signaling that influences cellular proliferation, growth, movement, synaptic stabilization, and apoptosis. ECM molecules, therefore, are prime candidates as contributors to the neural plasticity presumed to accompany memory consolidation.

The ECM network is composed of secreted glycoproteins and proteoglycans to which cells adhere. The brain ECM network consists predominantly of the proteins fibronectin, laminin, vitronectin, thrombospondin, tenascin, and collagen IV (Bosman & Stamenkovic, J Pathol, 200:423-28, 2003; Wright & Harding, Prog Neurobiol, 72:263- 93, 2004). Both fibronectin and laminin are glycoproteins that possess considerable neurite-promoting activity with respect to primary cultured neurons taken from mammalian embryonic brain.

The interaction among cells and ECM molecules is dependent upon various types of cell adhesion molecules (CAMs). These molecules are cell surface macromolecules that direct cell-to-cell and cell-to-ECM contacts by mediating the processes of adhesion, migration, neurite outgrowth, fasciculation, synaptogenesis, and intracellular signaling. The dynamic nature of the ECM and the impact of changes in the ECM on synaptic function are in large part dependent on the involvement of matrix metalloproteinases (MMPs).

MMPs comprise a family of zinc-dependent endo peptidases that includes over 25 distinct members (including, for example, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20). MMPs are critical to maintenance and restructuring of the ECM, where MMPs dynamically degrade and alter extracellular matrix (ECM) structure and function (Matrisian, 1992). In addition to degrading ECM proteins, MMPs can also cleave the extracellular domain of CAMs thus affecting communication between the ECM and pre- and post-synaptic neurons. MMPs have been associated with altered morphology and plasticity in multiple tissues (Vu and Werb, 2000; Sternlicht and Werb, 2001 ; Mott and Werb, 2004).

MMPs are implicated in remodeling and are positioned to globally affect cellular morphology, physiology and behavior (Sternlicht and Werb, Annu Rev Cell Dev Biol, 17:463-516, 2001). The recognition of MMPs as key enzymes in both normal and abnormal nervous system function is relatively recent. Both MMP-2 and -9 have been shown to be elevated in multiple sclerosis and stroke patients (Anthony et al., 1998), while increased latent hippocampal MMP-9 and decreased MMP-3 in Alzheimer's patients has led to speculation that a decrease in active MMP-9 could be involved in the deposition of beta-amyloid (Deb and Gottschall, 1996; Lim et al., 1997; Yoshiyama et al., 2000). Although globally expressed in the CNS, MMPs are enriched in hippocampi, and are important for hippocampal-mediated cognitive tasks. Research by Backstrom et al., suggests that MMPs are highly expressed in the hippocampus of Alzheimer's patients as compared with age-matched controls (Backstrom et al., J Neurosci, 16:7910-29, 1996). Szklarczyk et al. and Wright et al. report that changes in MMP function have been correlated with altered synaptic plasticity in the hippocampus (Szklarczyk et al., J Neurosci, 22:920-30, 2002; Wright et al., Brain Res, 963:252-61 , 2003), and research reported by Nakagami et al. suggests that experiments employing ECM degrading enzymes similar to MMPs indicate that elevated levels have a deleterious effect on hippocampal synaptic plasticity (Nakagami et al., J. Neurosci, 20:2003-10, 2000). According to particular aspects of the present Invention, inhibition of MMPs (e.g., MMP- 3 and/or MMP-9) in aged individuals has substantial utility to improve cognitive function: Previous studies by the present inventors involving habituation of the head- shake response in young adult rats have indicated that MMP-dependent synaptic remodeling is required for learning and memory consolidation, with the initial interpretation that high levels of MMPs are compatible with improved cognitive function and conversely low MMP levels may reflect poor cognition; induction of changes in brain matrix metalloproteinases-3 (MMP-3) and -9 (Meighan et al., Behav Brain /?es.,174(1):78-85; Nov. 1 , 2006, Epub 2006 Aug 17; incorporated herein by reference). In this previous study (Id), Western blots indicated significant elevations in the expression of matrix metalloproteinase-3 (MMP-3) in hippocampal, prefrontal and piriform cortices at a delay interval of 2 h, and in the prefrontal cortex at 24 h in habituated rats. Increases in active and pro MMP-9 activity were measured by zymography in the hippocampus of habituated rats over yoked controls (Id). Decreases in active MMP-9 activity were seen in the prefrontal cortex, and in pro MMP-9 in the piriform cortex, of habituated as compared with yoked control rats (Id). No changes in MMP-3 or MMP-9 were observed in the cerebellum, and no changes in MMP-2 were seen in any of the four structures examined (Id). Therefore, these prior results suggested that habituation of the HSR produced elevations in MMP-3 expression in three of the four structures examined, accompanied by increased MMP-9 activity in the hippocampus and decreases in the prefrontal cortex (Id).

Likewise, another prior publication by Applicants (J Neurochem., 96(5): 1227-41, March 2006; Epub 2006 Feb 8), involving rats learning the Morris water maze showed that inhibition of MMP activity with MMP-3 and -9 antisense oligonucleotides and/or MMP inhibitor FN-439 altered long-term potentiation and prevented acquisition in the Morris water maze. It is known that rats learning the Morris water maze exhibit hippocampal changes in synaptic morphology and physiology that manifest as altered synaptic efficacy, that the learning requires structural changes in the synapse, and multiple cell adhesion molecules appear to participate, and that the activity of these cell adhesion molecules is, in large part, dependent on their interaction with the extracellular matrix (ECM). In this prior study, therefore, Applicants had predicted that MMP function was critical for hippocampal-dependent learning, and as described in this prior publication (Id), it was observed that hippocampal MMP-3 and -9 increased transiently during water maze acquisition as assessed by western blotting and mRNA analysis. The ability of the NMDA receptor channel blocker MK801 to attenuate these changes indicated that the transient MMP changes were in large part dependent upon NMDA receptor activation. Furthermore, inhibition of MMP activity with MMP-3 and -9 antisense oligonucleotides and/or MMP inhibitor FN-439 altered long-term potentiation and prevented acquisition in the Morris water maze. The learning-dependent MMP alterations were shown to modify the stability of the actin-binding protein cortactin, which plays an essential role in regulating the dendritic cytoskeleton and synaptic efficiency. Together these results indicated that changes in MMP function are critical to synaptic plasticity and hippocampal-dependent learning. Applicants' original model thus envisioned that proteins of the extracellular matrix

(ECM) that lie in the synaptic cleft between pre- and post-synaptic elements act as a "glue" that holds the elements in an orientation that is optimal for synaptic communication, and MMPs serve to dissolve the glue by degrading the ECM. This is critical to learning-associated plasticity because in order for improved communication to result, a new "optimal orientation" of synaptic elements must be achieved that in turn underlies a new memory. As such, sufficiently high MMP levels were viewed as necessary to facilitate this process.

Surprisingly, . however and as presently disclosed herein, MMP . levels are substantially elevated in aged rats known to have cognitive deficits, and this observation has now led to a fundamental reworking of applicants' conceptual model of how MMPs impact synaptic function. Specifically, while MMPs may serve to 'dissolve' the ECM in facilitating learning-associated plasticity, the ECM must be reestablished so that new synaptic orientations (e.g., that underlie new memories) can be locked into place. Therefore, according to particular aspects of the present invention, and without being bound by any particular mechanism, increases in MMPs during learning- associated plasticity are transient— following a critical temporal pattern. Furthermore, according to additional aspects, conditions involving sustained markedly high levels of MMPs, as seen in aged individuals (e.g., rats), actually inhibit 'locking in' or reestablishment of optimal new synaptic orientations that are required for new learning to take place.

Specifically, and surprisingly as disclosed and shown in exemplary aspects herein: 1) aged rats not only have dramatically elevated MMPs in the hippocampus, but the levels of MMPs in individual rats are inversely correlated with cognitive ability;

2) aged rats have a depressed long-term potentiation (LTP) response in the hippocampus (LTP is an art-recognized measure of synaptic plasticity that has been positively correlated with memory storage); and 3) treating hippocampal slices taken from older rats (18-20 months) with broad spectrum MMP inhibitors (MMPi) significantly improves LTP.

Therefore, according to preferred aspects of the present invention, inhibition of

MMPs in aged individuals (e.g., rats) has substantial utility to improve cognitive function. In particular aspects, Inhibition of MMPs prevents hippocampal atrophy and amelioration of age-related cognitive deficits by inhibition of hippocampal MMPs in aged individuals.

Certain embodiments of the invention disclosed herein therefore relate to the use of MMP inhibitors in aged individuals to reverse cognitive decline. A number of different inhibitors have utility for this purpose. Prior clinical trials have tested the effectiveness of many MMP inhibitors as regulators of angiogenesis, and numerous MMP compounds have already passed FDA toxicology and therefore are suitable for the presently disclosed novel use. Exemplary MMP inhibitors encompasses compounds with varied substrate specificities and pharmacodynamic properties, such as blood-brain-barrier permeability. TABLE 1 shows a number of exemplary compounds having therapeutic utility according to particular aspects of the present invention.

The term "treating" refers to, and includes, reversing, alleviating, inhibiting the progress of, or preventing a disease, disorder or condition, or one or more symptoms thereof; and "treatment" and "therapeutically" refer to the act of treating, as defined herein.

A "therapeutically effective amount" is any amount of any of the compounds utilized in the course of practicing the invention provided herein that is sufficient to reverse, alleviate, inhibit the progress of, or prevent a disease, disorder or condition, or one or more symptoms thereof.

The methods of the invention include methods of treating a disease, condition or disorder of the peripheral or central nervous system in a mammal comprising the administration of a therapeutically effective amount of at least one compound that inhibits one or more MMPs (e.g., (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20 MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19 and MMP-20, etc.). Preferably, the disease treated is cognitive impairment (e.gr., cognitive decline in aged individuals). In particular aspects, MMP-3 and/or MMP- 9 is/are inhibited. In particular embodiments, at least MMP-3 is inhibited, along with one or more of the other MMPs. In certain embodiments, both MMP-3 and MMP-9 are inhibited. In certain embodiments, both MMP-3 and MMP-9 are inhibited, along with one or more of the other MMPs.

In particular aspects, the at least one compound is selected from the group consisting of the compounds in TABLE 1 herein, or a MMP-inhibiting derivative or analog thereof. Particular compounds of the present invention comprise asymmetric carbon atoms (e.g., optical or chiral centers) or double bonds, and the racemates, diasteriomers, geometric isomers and individual isomers, enantiamers (e.g., (R) or (S)), etc., are all intended, according to particular aspects of the present invention, to be encompassed within the scope of the present invention. Additionally, in particular embodiments, isotopic variations, whether radioactive (e.g., ³H, ¹²⁵I, ¹³¹I, ¹⁴C, ³²P₁ ¹¹¹In, ⁹⁰Y, etc.) or not, are likewise intended to be encompassed within the scope of the present invention. The compounds may have chiral centers and therefore may exist in different enantiomeric forms. In alternate aspects, the invention relates to all optical isomers, tautomers and stereoisomers of the MMP-inhibiting compounds and mixtures thereof, and to methods comprising the administration of pharmaceutically acceptable non-toxic salts (e.g., acid or basic addition salts) of these compounds (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (e.g., 1 ,1'-methylene-bis-(2- hydroxy-3-naphthoate)); or potassium, sodium, calcium and magnesium, ammonium, N-methylglucamine-(meglumine), and lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines).

The present invention also relates to a pharmaceutical compositions for the treatment of a disease, condition or disorder of the peripheral or central nervous system, wherein the disease, condition or disorder is Alzheimer's disease, stroke/cerebral ischemia, head trauma, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, migraine, cerebral amyloid angiopathy, AIDS, age-related cognitive decline; mild cognitive impairment or a prion disease. Preferably, the pharmaceutical composition is for the treatment of cognitive impairment (e.g., cognitive decline in aged individuals).

Particular aspects encompasses pharmaceutical compositions comprising a drug or prodrug of one or more compounds selected from the group consisting of the exemplary compounds in TABLE 1 herein, or a MMP-inhibiting derivative or analog thereof. Additional aspects encompasses methods of treating or preventing disorders that can be treated or prevented by the inhibition of matrix metalloproteinases comprising administering of such drugs or prodrugs. The exemplary compounds in TABLE 1 having, for example, a free amino, amido, hydroxy or carboxylic group can be converted into a prodrug. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to, for example, such free amino, hydroxy or carboxylic acid groups of the compounds. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3- methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Exemplary prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents through the carbonyl carbon prodrug side chain.

One of ordinary skill in the art will appreciate that when using the methods of the invention in the treatment of a specific disease (e.g., age-related cognitive decline or impairment), that the methods of the invention may be combined with various existing therapeutic methods and agents used for that disease; that is, combination therapies, or using combination pharmaceutical compositions. For example, the MMP inhibiting compound(s) may be used in conjunction with standard non-steroidal anti-inflammatory drug (NSAID¹S; such as piroxicam, diclofenac), a propionic acid (e.g, naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen), a fenamate (e.g., mefenamic acid, indomethacin, sulindac, apazone), a pyrazolone (e.g., phenylbutazone), a salicylate (e.g., aspirin), an analgesic or intraarticular therapy (e.g., a corticosteroid), and a hyaluronic acid (e.g., hyalgan and synvisc), an immune suppressant (e.g., cyclosporin, interferon, etc., e.g., in organ transplant therapy), a TNF-.alpha. inhibitor (e.g., Enbrel.RTM.), low dose methotrexate, lefunimide, hydroxychloroquine, d-penicilamine, auranofin, parenteral gold, oral gold, etc.

Additionally, when using the methods of the invention in the treatment of a specific disease (e.g., age-related cognitive decline or impairment), the MMP inhibiting compound(s) may also be used in conjunction with a CNS agent or agents, such as an antidepressant [e.g., sertraline, fluoxetine, paroxetine, etc.), an anti-Parkinsonian drug (e.g., deprenyl, L-dopa, requip, miratex, etc.), a MAOB inhibitor (e.g., selegine, rasagiline, etc.), a COMP inhibitor (e.g., tolcapone (i.e., Tasmar)) an A-2 inhibitor, a dopamine reuptake inhibitor, an NMDA antagonist, a nicotine agonist, a dopamine agonist, an inhibitor of neuronal nitric oxide synthase, an anti-Alzheimer's drug, an acetylcholinesterase inhibitor (e.g., metrifonate, donepezil (i.e., Aricept), Exelon (i.e., ENA 713 or rivastigmine), etc.), tetrahydroaminoacridine (i.e., Tacrine, Cognex, or THA), a COX-1 or COX-2 inhibitor (e.g., celecoxib (i.e., Celebrex), rofecoxib (i.e., Vioxx), etc.), propentofylline, an anti-stroke medication, an NR2B selective antagonist, a glycine site antagonist, a neutrophil inhibitory factor (NIF), etc.

Additionally, when using the methods of the invention in the treatment of a specific disease (e.g., age-related cognitive decline or impairment), the MMP inhibiting compound(s) may also be used in conjunction with an estrogen, a selective estrogen modulator (e.g., such as estrogen, raloxifene, tamoxifene, droloxifene, lasofoxifene, etc), an agent that results in reduction of A. beta.1-40/1 -42 (e.g., an amyloid aggregation inhibitor, a secretase inhibitor, etc.), an osteoporosis agents (e.g., droloxifene or fosomax), immunosuppressant agents (e.g., FK-506 and rapamycin), an anticancer agent (e.g., endostatin and angiostatin), a cytotoxic drug (e.g., adriamycin, dauπomycin, cis-platinum, etoposide, taxol, taxotere), or an alkaloid (e.g., vincristine), an antimetabolite (e.g., methotrexate, a cardiovascular agent (e.g., calcium channel blockers), a lipid lowering agent (e.g., a statin, a fibrate, a beta-blocker, an ACE inhibitor, an angiotensin-2 receptor antagonist or a platelet aggregation inhibitor).

Table 1. Exemplary Matrix Metalloproteinase (MMP) Inhibitors (e.g., obtainable from EMD Biosciences).

MMP-9/MMP-13 444253 IC₅₀ = 1.9 nM for MMP-9 and 1.3 Inhibitor Il nM for MMP-13. Also inhibits MMP- 1 (IC₅₀ = 24 nM), MMP-3 (IC₅₀ = 18 nM), and MMP-7 (IC₅₀ = 230 nM).

N-Hydroxy-1-(4-methoxyphenyl)sulfonyl-4- benzyloxycarbonylpiperazine-2-carboxamide

Trocade See Marion Flipo et al., "A library of novel hydroxamic acids targeting the metallo-protease family: Design, parallel synthesis and screening," Bioorganic & Medicinal Chemistry 15, pp. 63-76 (2007) incorporated herein by reference in its entirety.



Exemplary preferred embodiments:

Particular aspects provide methods for treating cognitive impairment, comprising administration to a subject in need thereof a therapeutically effective amount of at least one matrix metalloproteinase (MMP) inhibitor sufficient to provide for at least one of precluding, alleviating, reversing, or inhibiting cognitive impairment characterized by persistent or sustained elevated expression and/or activity of at least one MMP. In particular embodiments, the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least one MMP having the characteristic elevated expression or activity. In additional embodiments, the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least two MMPs having the characteristic elevated expression or activity. In certain embodiments, the at least one matrix metalloproteinase (MMP) inhibitor is MMP-specific or substantially specific to a particular MMP or inhibits a limited number of MMPs (e.g., from one to two MMPs, from one to three MMPs, or from about one to about four MMPs). In certain embodiments the at least one matrix metalloproteinase (MMP) inhibitor is a broad spectrum MMP inhibitor inhibiting at least three, or at least 4 MMPs (e.g., having the characteristic elevated expression or activity). In certain aspects, the cognitive impairment is characterized by sustained elevated MMP-3 expression and/or activity, where the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least matrix metalloproteinase 3 (MMP-3).

In particular embodiments, the cognitive impairment is further characterized by sustained elevated expression and/or activity of at least one additional MMP, where the at least one matrix metalloproteinase (MMP) inhibitor is suitable to additionally inhibit the additional matrix metalloproteinase (MMP). In certain embodiments, the at least one additional matrix metalloproteinase (MMP) comprises MMP-9. In particular exemplary embodiments of the methods, the at least one metalloproteinase (MMP) inhibitor comprises a hydroxymate moiety. In certain aspects, for example, the at least one metalloproteinase (MMP) inhibitor comprises a zinc-binding hydroxamate moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase. In additional aspects, the at least one metalloproteinase (MMP) inhibitor comprises a non-hydroxamate zinc-binding moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase. In certain aspects, the at least one matrix metalloproteinase (MMP) inhibitor is at least one selected from the group of compounds of Table 1 disclosed herein. In certain embodiments, the at least one matrix metalloproteinase (MMP) inhibitor comprises 4-Abz-Gly-Pro-D- Leu-D-Ala-NH-OH or a derivative thereof, wherein Abz is aminobenzoyl.

In particular embodiments, treating comprises prophylactic administration. In certain embodiments, treating comprises administration to alleviate cognitive impairment. In particular aspects, treating comprises administration to inhibit cognitive impairment. In particular embodiments, treating comprises administration to reverse cognitive impairment.

In certain aspects, the cognitive impairment characterized by sustained elevated MMP expression and/or activity is age-related cognitive impairment. In certain embodiments, the subject is determined to have age-related cognitive impairment characterized by sustained elevated MMP expression. In particular aspects, the subject is determined to have age-related cognitive decline, dementia or Alzheimer's disease. Additional embodiments of the methods further comprise combination therapy, wherein the at least one matrix metalloproteinase (MMP) inhibitor is used in conjunction with at least one additional therapeutic agent. In certain aspects, the at least one additional therapeutic agent is selected from the group consisting of: standard non-steroidal anti-inflammatory drugs (NSAID¹S), piroxicam, diclofenac; a propionic acid, naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen; a fenamate, mefenamic acid, indomethacin, sulindac, apazone; a pyrazolone, phenylbutazone; a salicylate, aspirin; an analgesic or intraarticular therapy, a corticosteroid; a hyaluronic acid, hyalgan, synvisc; an immune suppressant, cyclosporine, interferon; a TNF-.alpha. inhibitor, Enbrel™; low dose methotrexate, lefunimide, hydroxychloroquine, d-penicilamine, auranofin, parenteral gold and oral gold.

In other aspects, the at least one additional therapeutic agent is selected from the CNS agent group consisting of: an antidepressant, sertraline, fluoxetine, paroxetine; an anti-Parkinsonian drug; deprenyl, L-dopa, requip, miratex; a MAOB inhibitor, selegine, rasagiline; a COMP inhibitor, tolcapone, Tasmar; an A-2 inhibitor, a dopamine reuptake inhibitor, an NMDA antagonist, a nicotine agonist, a dopamine agonist, an inhibitor of neuronal nitric oxide synthase, an anti-Alzheimer's drug; an acetylcholinesterase inhibitor, metrifonate, donepezil, Aricept, Exelon, ENA 713 or rivastigmine; tetrahydroaminoacridine, Tacrine, Cognex, or THA; a COX-1 or COX-2 inhibitor, celecoxib, Celebrex, rofecoxib, Vioxx; propentofylline, an anti-stroke medication, an NR2B selective antagonist, a glycine site antagonist, and a neutrophil inhibitory factor (NIF). In other embodiments, the at least one additional therapeutic agent is selected from the group consisting of: an estrogen; a selective estrogen modulator, estrogen, raloxifene, tamoxifene, droloxifene, lasofoxifene; an agent that results in reduction of A. beta.1-40/1 -42, an amyloid aggregation inhibitor, a secretase inhibitor; an osteoporosis agent, droloxifene, fosomax; immunosuppressant agents, FK-506, rapamycin; an anticancer agent, endostatin, angiostatin; a cytotoxic drug, adriamycin, daunomycin, cis- platinum, etoposide, taxol, taxotere; an alkaloid, vincristine; an antimetabolite, methotrexate; a cardiovascular agent, calcium channel blockers; a lipid lowering agent, a statin; a fibrate, a beta-blocker, an ACE inhibitor, an angiotensin-2 receptor antagonist , and a platelet aggregation inhibitor.

ADMINISTRATION and DOSAGE: Preferred compounds (e.g., the compounds in TABLE 1 herein, or a

MMP-inhibiting derivative thereof) include those inhibitors that possess potent activity against MMP-3 and/or MMP-9 (preferably an IC₅₀ of less than 500 nM, more preferably 100 nM, most preferably 50 nM) preferably wherein said MMP-3 and/or MMP-9 inhibitory activity is selective activity for MMP-3 and/or MMP-9.

For administration to mammals, including humans, in accordance with the methods of treatment of the present invention, for the treatment of a disorder, conditions or disease of the peripheral or central nervous system, a variety of conventional routes may be used including oral, parenteral (e.g., intravenous, intramuscular or subcutaneous), buccal, anal and topical. In general, the compounds of the invention will be administered at dosages between about 0.1 and 25 mg/kg body weight of the subject to be treated per day, preferably from about 0.3 to 5 mg/kg. Preferably the active compound will be administered orally or parenterally. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The compounds of the present invention can be administered in a wide variety of different dosage forms, in general, the therapeutically effective compounds of this invention are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelation and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof. In the case of animals, they are advantageously contained in an animal feed or drinking water in a concentration of 5-5,000 ppm, preferably 25 to 500 ppm.

For parenteral administration (intramuscular, intraperitoneal, subcutaneous and intravenous use) in accordance with the present invention, a sterile injectable solution of the active ingredient is usually prepared. Solutions of a therapeutic compound of the present invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably adjusted and buffered, preferably at a pH of greater than 8, if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and. subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. In the case of animals, compounds can be administered intramuscularly or subcutaneously at dosage levels of about 0.1 to 50 mg/kg/day, advantageously 0.2 to 10 mg/kg/day given in a single dose or up to 3 divided doses.

For the methods of the present invention, the active compounds herein disclosed may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation, the active compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

The following materials and methods have utility in the Examples that follow hereunder.

Behavioral methods. The protocols used in these experiments are approved by the Washington State University Institutional Animal Care and Use Committee and conform to the guidelines for the care and use of laboratory animals as required by the National Institutes of Health. Male Sprague-Dawley rats (3 months old, Charles Rivers-derived) are adapted to a 12-hour light/dark cycle and were permitted access to water and Purina laboratory rat chow ad libitum. Each animal fs handled for five min per day beginning two days before the initiation of maze training.

The water maze protocol has previously been described in detail (Wright et al. 1999). Briefly, it consists of a 1.6 m diameter x 0.6 m-tall galvanized, cylindrical tank painted black and filled to a depth of 30 cm with 26-28°C water. Geometrical visual cues are placed on three walls of the test room. The experimenter stands in a consistent location and serves as a visual cue against the fourth wall. Latency and path distance to a submerged, hidden pedestal are measured using a video tracking system and computer tracking software (Chromotrak; San Diego Instruments, San Diego, CA).

For each subject the pedestal location is constant throughout training. The entry point into the maze is randomly varied with each trial among four possible entry sites (N₁ S, E or W). The subjects are placed into the maze facing the pool wall. Subjects are allowed five trials per day, 120 sec per trial, to find the hidden platform. If the subject finds the platform within the 120 sec it is given a 30 sec rest period on the platform between trials. If the pedestal is not located within the time allotted, the subject is placed onto the platform and allowed 30 sec until the next trial.

Animals are assigned randomly to groups (n=8 per group), which determines the number of days the subjects are trained in the maze. Seven groups of animals are trained in the Water maze for 1, 2, 3, 4, or 5 days. Additional rats are utilized for water maze yoked animals and swim for an equal duration in the maze in a darkened room without an escape platform. A final group comprises home cage control animals that are naϊve to the maze. At the end of each test day, the animal is dried with a towel, placed under a 100 W warming lamp for 15 min, and then returned to its home cage. To assess potential drug-associated motor impairment, animals are subjected to a standard motor battery, which includes righting reflex, tilted platform test and rope balance. In addition, swim speed may be calculated from the measured maze times and distances.

Tissue preparation. At a specified time following the final water maze trial of each test day, rats are decapitated, and the hippocampus from each hemisphere is quickly dissected on ice, immediately frozen in liquid nitrogen and stored at -80⁰C until all sample groups are collected. One hippocampus from each animal is analyzed for MMP protein levels and the other for MMP mRNA expression (see RT-PCR analysis methods).

Tissues for protein analysis are weighed and immediately homogenized on ice in a volume of homogenization buffer (5OmM Tris HCI pH 7.6, 150 mM NaCI, 5 mM CaCI₂, 0.05% Brij 35, 0.02% NaN₃) to give a final sample concentration of 1mg/ml. Homogenates are centrifuged at 12,000 x g for 5 min, 4°C and the supernatant fraction may be recovered for analysis by immunoblotting. Western immunoblotting. Supernatants are mixed 1:1 with 2x Laemelli sample buffer plus β-mercaptoethanol. Samples are subjected to SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. Following transfer, membranes are pre-blocked in 4% milk/ TBS prior to the addition of primary antibody. Membranes are incubated in primary antibody overnight at 4°C [1:2000, MMP-9 (Abeam, Cambridge, MA); 1:2000, MMP-3 (RDI, Flanders, NJ); 1:1000, MMP-2 (Chemicon, Temecula, CA); 1:2000 cortactin (Upstate, Charlottesville, VA)]. After rinsing, blots are incubated for 2 hours with a 1 :10,000 dilution of HRP-conjugated secondary antibody and rinsed again in TBS/TTBS. Visualization is achieved with Pierce SuperSignal and subsequent exposure to Kodak X-Omat Blue film. Signal intensity per volume may be quantitated using TotalLab Image Analysis software.

MMP-3 immunohistochemistry. In general, three rats are trained for one day in the water maze. Three home cage control animals are naϊve to the maze. Four hours following the final trial, each rat is anesthetized and perfused through the left ventricle of the heart with 0.1 M PBS followed by 10% formalin. The brain is removed and placed in cold 10% formalin for 24 hours. The brains are then blocked and frozen in preparation for sectioning.

Subsequent to cryostat sectioning, coronal slices (30 μm) are subjected to immunohistochemical analysis using standard methods, briefly described here. Free-floating sections are rinsed in 0.4 M PBS and incubated in 50% ETOH for 30 min. After rinsing in 0.4 M PBS, the slices are incubated in 10% normal horse serum for 30 min. Sections are then incubated overnight in anti-MMP-3 (1:1000) (control sections were incubated in the absence of primary antibody), washed in PBS and placed in appropriate secondary antibody overnight. MMPs are visualized using ExtraAvidin Peroxidase Conjugate and Ni-DAB. Sections are then mounted on gelatinized slides, taken through ETOH fixation and cover-slipped. Hippocampal sections may be visualized using standard light microscopy and digitally photographed. MMP-3 and MMP-9 transcript analysis by RT-PCR. Total RNA from hippocampal tissue may be extracted using TRIZOL (Invitrogen, California, USA). 1 μg of total RNA is incubated at 70⁰C for ten minutes with oligo-dT primers and then immediately chilled on ice. The cDNA synthesis reaction mixture of 10X buffer (200 mM Tris HCI (pH 8.4) and 500 mM KCI), 10 mM dNTPs, 25 mM MgCI₂, 100 m M DTT, Ribonuclease inhibitor and 50 units Superscript Il reverse transcriptase (all from Invitrogen, California, USA, used according to manufacturer's direction) is assembled on ice and the reaction is allowed to proceed at 42°C for 50 min. The cDNA synthesis is terminated at 70⁰C for 15 min and then chilled on ice. 1 μl of the total 20 μl RT reaction may be used as template DNA for

PCR. The amount of template is chosen to reside in the linear portion of a preliminary amplification curve that is derived by serially diluting the template. PCR is performed in a 25 μl reaction mixture with 1X buffer (200 mM Tris HCI (pH8.4), 500 mM KCI), dNTPs (0.2 mM each), MgCI₂ (1.5 mM), forward and reverse primer mix (200 μM each), and platinum Taq DNA polymerase (1.0 unit) in autoclaved distilled water. Amplification is performed for 30-40 cycles (denaturation at 94⁰C for 15 sec, annealing at 55°C-60°C for 30 sec and extension at 72°C for 30 sec. To normalize RT-PCR results, PCR using GAPDH specific primers may be performed. Sequences for sense primers for MMP-3, MMP-9 and GAPDH include S'-TTCTCCAGGATCTCTGAAGGAGAGG-S' (SEQ ID NO: 1), 5'- AAATGTGGGTGTACACAGGC-S' (SEQ ID NO:2) and 5'- CTGGAGAAACCTGCCAAGTATGAT-3' (SEQ ID NO:3) respectively. Antisense sequences include 5'-ATTTGGTGGGTACCACGAGGA-S' (SEQ ID NO:4), δ'-TTCACCCGGTTGTGGAAACT-S' (SEQ ID NO:5) and 5'- TTCTTACTCCTT GGAGGCCATGTA-S¹ (SEQ ID NO:6), respectively. Products of 391, 309 and 267 base pairs are predicted for MMP-3, MMP-9 and GAPDH, respectively. 10 μl of 25 μl total PCR reaction may be analyzed in a 2% agarose gel in 1X TAE (40 mM Tris-acetate, 1 mM EDTA). The DNAs are visualized by ethidium bromide staining and ultraviolet illumination. Gels may be digitally photographed, scanned, and quantitated by densitometry (Totallab).

Intracerehroventricular cannula placement. Male Sprague-Dawley rats are anesthetized and fitted with a unilateral i.c.v. guide cannula as previously described (Pederson et al. 1998). After retraction of the scalp, a hole is drilled through the skull 1 mm posterior to Bregma and 1.5 mm lateral to midline and a PE-60 guide cannula is inserted and held in place using holding screws and dental cement. The scalp is sutured and animals are allowed to recover for 10 days prior to drug administration and behavioral testing in the water maze.

MMP inhibitor administration in vivo. The protocol for in vivo MMP inhibition is similar to that of Reeves et al. (Reeves et al. 2003); however, a modified injection volume is used. Ten minutes prior to behavioral testing rats receive intracerebroventricular (i.c.v.) infusions of MMP inhibitor FN-439 (Sigma, St. Louis, MO; 7.2 mM stock in aCSF, 10 μl over 5 mfn) or an equal volume of aCSF. All rats receive a second injection (identical to their first injection) three hours post-training.

MMP antisense administration in vivo. Rats (n=4) are infused bilaterally with 100 nl of an antisense oligodeoxynucleotide (ODN) mixture containing 1 nmole each of an ODN for MMP-3 (5'- C*A*GGACTGGGAGCCCTTTC*A*T-3' (SEQ ID NO:7); *= phosphothiorate linkage) and MMP-9 (5'-C*A*GGGGCTGCCAGGGACTC*A*T-3'; (SEQ ID NO:8) *= phosphothiorate linkage) or control combination of sense ODNs for MMP-3 (5'-C*A*GGACTGGGAGCCCπTC*A*T-3' (SEQ ID NO:9); *= phosphothiorate linkage) and MMP-9 (5'-

A*T*GAGTCCCTGGCAGCCCC*T*G-3' (SEQ ID NO:10); *= phosphothiorate linkage). Infusions are carried out over a 5 min period at a rate of 20 nL/m using a precision nanoliter syringe pump. Twelve hours after ODN infusion, water maze training is initiated as described above.

The only deviation from the standard training protocol is that training consists of 15 trials for one day only. The purpose of limiting the training to one day is to coordinate training with the established time course of action of the antisense ODNs. Preliminary studies demonstrate that the mixture of MMP-3 and MMP-9 antisense ODNs have maximal effects on reducing MMP- 3 protein levels in the hippocampus 12 hr after infusion, that this effect is sustained for 24 hr, but reversed by 48 hr.

LTP slice preparation. Rats are anesthetized with Halothane (Halocarbon Laboratories, River Edge, NJ), decapitated and the brain rapidly removed. After the brain is removed, it is immediately placed into ice-chilled, oxygenated artificial cerebrospinal fluid (aCSF) that contained 124 mM NaCI, 3 mM KCI₁ 1.24 mM Na₂PO₄, 1.3 mM MgSO₄, 2.0 mM CaCI₂, 26 mM NaHCO₃, and 10 mM D-glucose for approximately 30 sec. The hemispheres are separated by a mid sagittal cut and the hippocampus is removed from the right hemisphere using a custom fashioned wire loop instrument. Slices (400 μm thick) from the middle third portion of the hippocampus are prepared using a Mcllwain tissue chopper (Brinkmann/Gomshall, Surrey, Great Britain) and transferred to a gassed (95% O₂/5% CO2) incubation chamber containing aCSF, where they are maintained for at least 1 h at 22~23°C. Single slices are then transferred to a perfusion-recording chamber and stabilized on the chamber floor (coated with Sylgard, Dow Corning, Midland, Ml) by platinum wires. Slices are continuously superfused with gassed aCSF (30-31⁰C) at a rate of 1-1.5 ml/min via a peristaltic pump (Rainen Rabbit-Plus, Woburn, MA). LTP slice recording. Extracellular recordings from the CA1 stratum radiatum layer are obtained using glass micropipettes filled with 0.15 M NaCI yielding a resistance of 2 to 3 MΩ. Orthodromic activation of the Schaffer collaterals within the CA1 region may be accomplished using concentric bipolar stimulating electrodes (Rhodes Medical Instruments, Inc., Woodland Hills, CA) positioned adjacent to the recording electrodes in the CA1 stratum radiatum layer. Test stimuli (0.1 ms, 0.1 Hz) are delivered using a Grass stimulator (Model S88, Quincy, MA) to elicit field excitatory postsynaptic potential (fEPSP) responses. The intensity of stimulation is adjusted in each case to produce a dendritic field potential that is 50-60% of the maximum spike-free response (1-1.5 mV). Following 10 min of pre-tetanus in the presence of FN-439 (180 μM, Sigma Chemical, St. Louis, MO) or MMP inhibitor cocktail (MMPi) (MMP-2/MMP-3 Inhibitor I, 9 nM in aCSF and MMP- 2/MMP-9 Inhibitor IV₁ 10 μM in aCSF), Calbiochem, San Diego, CA), high frequency stimulation is used to induce LTP. This is accomplished by the application of five theta burst stimulations (TBS) separated by 200 ms. Each burst contains four pulses at 100 Hz. This train may then be repeated three times with an inter-train interval of 10 sec.

LTP may be defined as a greater than 20% increase in fEPSP slope measured at 30 min post-tetanic stimulation. Extracellular signals are amplified (gain 1000X) and filtered (1 kHz) using an A-M Systems amplifier (Model 1800, Newport, WA). Data are digitized and analyzed using a computer-interfaced PowerLab/400 (Dover, NH). Three successive peak slope measurements of the initial phase of fEPSP response may be averaged and recorded. In control experiments, baseline fEPSP is recorded in the presence of FN-439 over 60 min.

MK801 Administration. Rats (n=5) receive a daily injection of MK-801 (0.1 mg/kg, i.p., Calbiochem, San Diego, CA) 15 min prior to water maze training. Four hours following the final trial on day two of training, rats are sacrificed and hippocampal tissue taken for MMP and cortactin protein analysis by Western blotting.

Data analyses. MMP data may be expressed as percent home cage control +/- SEM, which provided an internal standard that made gel-to-gel comparisons possible. Training data are expressed as the mean daily latency for five trails per group +/- SEM (n=8/group). One-way ANOVA and student t- test or Welch's corrected t-test were used where appropriate. LTP data are recorded at 1 min intervals, and for each slice fEPSP slopes are averaged over a 10 min baseline-recording period. Each fEPSP is normalized relative to the average baseline response and expressed as percent change from baseline.

Example 1

(Additional Materials and Methods used in the Examples herein)

Generation of Hippocampal Slices. Rats were anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ, USA), decapitated and the brain was rapidly removed. The brain was immediately placed into ice- chilled, oxygenated aCSF for approximately 30 s. The hemispheres were separated by a mid-sagittal cut and the hippocampus was removed from the right hemisphere using a custom-built wire loop instrument. Slices (400 μm thick) from the middle third portion of the hippocampus were prepared using a Mcllwain tissue chopper (Brinkmann, Gomshall, UK) and transferred to a gassed (95% O₂ / 5% CO₂) incubation chamber containing aCSF, where they were maintained for at least 1 h at 22— 23°C. Single slices were then transferred to a perfusion-recording chamber and stabilized on the chamber floor (coated with Sylgard; Dow Corning, Midland, Ml, USA) by platinum wires. Slices were continuously superfused with gassed aCSF (30-31⁰C) at a rate of 1—1.5 mL/min via a peristaltic pump (Rainen Rabbit-Plus, Woburn, MA, USA).

Electrophysiology. Extracellular recordings from the CA1b stratum radiatum layer were obtained using glass micropipettes filled with 0.15 M

NaCI, yielding a resistance of 2-3 MΩ. Orthodromic activation of the Schaffer collaterals within the CA1c was accomplished using concentric bipolar stimulating electrodes (Rhodes Medical Instruments, Inc., Woodland Hills, CA, USA) positioned adjacent to the recording electrodes in the CA1c stratum radiatum layer. Test stimuli (0.1 ms, 0.1 Hz) were delivered using a stimulator (Model S88; Grass, Quincy, MA, USA) to elicit field excitatory postsynaptic potential (fEPSP) responses. The duration of stimulation was adjusted in each case to produce a dendritic field potential that was 50-60% of the maximum spike-free response (.08-.12 ms).

LTP was induced by theta patterned high-frequency stimulation. This was accomplished by the application of 4 trains of theta burst stimulations (TBSs) with an inter-train interval of 10 s. Each train was comprised of 6 bursts of four 100Hz, .35ms pulses, separated by 200ms.

Extracellular signals were amplified (gain 1000x) and filtered (1 kHz) using an amplifier (Model 1800; A-M Systems, Newport, WA, USA). Data were digitized and analyzed using a computer interfaced PowerLab/400 (ADInstruments Inc., Dover, NH, USA). Circular water maze training. Male Fisher-344 rats (22 months old, Charles Rivers-derived) were adapted to a 12-hour light/dark cycle and were permitted access to water and Purina laboratory rat chow ad libitum. Each animal was handled for five min per day beginning two days before the initiation of maze training. The water maze has previously been described in detail (Wright et al., 1999). Briefly, it consisted of a 1.6m diameter x 0.6m-tall galvanized, cylindrical tank painted black and filled to a depth of 30 cm with 26-28⁰C water. Geometrical visual cues were placed on three walls of the test room. The experimenter stood in a consistent location and served as a visual cue against the fourth wall. Latency and path distance to a submerged, hidden pedestal were measured using a video tracking system and computer tracking software (Chromotrak; San Diego Instruments, San Diego, CA).

For each subject the pedestal location was constant throughout training. The entry point into the maze was randomly varied with each trial among four possible entry sites (N, S, E or W). The subjects were placed into the maze facing the pool wall. Subjects were allowed four trials per day, 120 s per trial, to find the hidden platform. If the subject found the platform within the 120 s it was given a 30 s rest period on the platform. If the pedestal was not located within the time allotted, the subject was placed onto the platform and allowed 30 s until the next trial. Following each trial, the rat was removed and placed into a holding cage for 6 minutes subsequent to its next trial (6 minute intertrial interval). At the end of each test day, the animal was dried with a towel and placed under a 100 W warming lamp for 15 min and then returned to its home cage. For assessment of the effect of the MMP inhibitor (FN-439) on water maze performance, rats received i.p. injections (10 mg FN439 in 2.85 mis saline; 100 ul/100 g bodyweight) 2 hours prior to each day's training session. Control rats received vehicles (saline) injections 2 hours prior to water maze training. Western immυnoblotting. Supematants were mixed 1:1 with 2x Laemelli sample buffer plus β-mercaptoethanol. Samples were subjected to SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. Following transfer, membranes were pre-blocked in 5% milk/ TBS prior to the addition of primary antibody. Membranes were incubated in primary antibody overnight at 4° C, MMP-3 (RDI, Flanders, NJ) 1:1000, TIMP-2 (Chemicon, Temecula, CA) 1:1000, cortactin (Upstate, Charlottesville, VA)] 1:1000. After rinsing with tris-buffered saline (TBS) and tween-20 + tris-buffered saline (TTBS), blots were incubated for 2 hours with a 1:10,000 dilution of HRP- conjugated secondary antibody and rinsed again in TBS/TTBS. Visualization was achieved with Pierce SuperSignal and subsequent exposure to Kodak X- Omat Blue film. Signal intensity per volume was quantitated using TotalLab Image Analysis software.

Data analysis. Three successive fEPSP amplitude measurements were averaged and recorded. LTP and LTD time-courses were expressed as the percent of the mean baseline response. Two-factor ANOVA, area under the curve, student T-tests, and regression analyses were performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego California USA). Two-factor ANOVA performed with NCSS (Number Crunchers Statistical Systems, Kaysville UT). Specific analyses indicated within the figure legends. Prior to hypothesis testing, data tested for normality and equal-variance with the Omnibus Normality of Residuals and the Modified-Levene Equal-Variance tests respectively (NCSS). A test for interaction between factors was included for each multi-factor ANOVA. In each instance, the analysis failed to indicate a significant interaction between the tested factors. Because there were only two levels of treatment factor, a significant result was not followed by multiple comparisons testing.

Example 2 (Effects of age on and LTP and fEPSP characteristics from Schaffer collaterials/CAI field recordings were investigated)

Hippocampal slices generated from 3-month (young) and 22-month (aged) Sprague Dawley rats were analyzed for differences in evoked field responses during baseline and potentiated conditions. In accordance with previously published findings (Tombaugh, Rowe et al. 2002), slices generated from aged rats had a significant deficit in magnitude of theta burst stimulated LTP during induction and early maintenance phases compared to younger animals (two factor ANOVA; p<.01) (FIGURES 1A, B).

In situations where an augmented synaptic response is elicited, by either increased magnitude of stimulation or by synaptic potentiation, it is typical for the fEPSP peak to experience a leftward shift (i.e., peak values are achieved more rapidly than basal level responses). This phenomenon represents a change in the temporal efficiency in the interval of time between presynaptic depolarization (with concomitant neurotransmitter release) and maximum post-synaptic responsiveness. Quantification of this relationship between pre and post synaptic elements can be performed by measuring the latency from the peak of the presynaptic fiber volley to the peak of fEPSP (peak-to-peak latency). In accordance with this phenomenon, slices generated from young rats experience a significant reduction of peak-to-peak latency (i.e., increased temporal efficiency) upon tetanization which persists through early maintenance (t=25min -30min post tetanus) (FIGURE 1C). Conversely, tetanus induced modifications to peak-to-peak latency is relatively absent in slices generated from aged rats (Aspin-Welch test; p<.001). This is particularly true during early maintenance where peak-to- peak latency is not significantly greater than pre-tetanus levels (one sample t- test; p>.05). Interestingly, however, a comparison of baseline peak-to-peak latencies from both young and aged rats reveals a significantly reduced peak- to-peak latency in evoked field potentials from aged rats as compared to younger counterparts (FIGURE 1 D). The divergence in the temporal efficiency between young and aged animals was not due to difference in fEPSP amplitudes as there was no statistical difference in baseline amplitudes between the two groups.

Example 3

(Aged rats were shown to have chronic elevations of hippocampal MMPs)

Rats learning a hippocampal dependent task experience significant increases in hippocampal MMP-3 levels during task acquisition (Meighan, Meighan et al. 2006). It was demonstrated that these MMP alterations are necessary for water maze learning and synaptic plasticity {Id). Classically, MMP expression/activity within the CNS has been linked to pathological situations, such as ischemia (Lee, Tsuji et al. 2004), and tissue trauma (Phillips and Reeves 2001). A feature of MMP regulation during these conditions is that MMPs experience a persistent increase for days after initial insult. These seemingly divergent roles for MMPs led us to examine if there is evidence for a functional distinction between pathologically associated increases and the increases observed during learning. An observation, as disclosed herein, which may distinguish between these differences is that learning associated MMP-3 increases return to basal levels within 24 hours following training (FIGURE 2A). Therefore a potentially important characteristic of learning associated MMP increases is that MMP upregulation during these conditions are transient; contrasting chronic elevations seen in pathologically associated conditions. This suggested to applicants that learning associated MMP elevations are perhaps only appropriate during a discrete phase of learning associated synaptic remodeling. Particular literature documents MMP dysregulation in tissue aging (particularly in skin, and cardiac). Considering the importance of MMPs to hippocampal plasticity, that aged rats exhibit deficits to long term potentiation, and that MMPs are dysregulated in multiple tissues during aging, the present applicants conceived that abnormalities in the expression pattern of MMPs hippocampal tissue in aged rats are present. In contrast to the low levels found in young-adult rats, our assessment of hippocampal tissue from aged- adult rats uncovered chronic basal elevations in MMP-3 levels (FIGURE 2B). The increased MMP-3 levels are also significantly present in TIMP- sequestered form (FIGURE 2C).

Example 4

(MMP inhibitors facilitated LTP in hippocampal slices from aged rats)

Following applicants' initial observations, there were two competing possibilities which could potentially explain the functional consequences of increased hippocampal MMP expression in aged rodents.

One possibility is that age-linked MMP increases attend to compensatory function; this possibility seems reasonable considering MMPs importance in synaptic plasticity. A logical consequent to this premise would be that increased MMP activity is beneficial in helping preserve plasticity of hippocampal networks.

A second contending possibility is that increased MMPs are a significant source of burden placed on plasticity critical mechanisms. A consequent to this premise is that increased MMP activity is detrimental to processes underlying hippocampal plasticity.

Applicants conceived that if the former premise were true (i.e., MMPs are serving compensatory function in hippocampal aging) inhibition of MMPs should be detrimental to hippocampal plasticity. Conversely, if the latter premise were true (i.e., applicants conveived that MMPs are a burden to processes underlying hippocampal plasticity), MMP inhibition would be predicted to enhance plasticity dependent processes. Applicants tested these contending hypotheses by observing the impact of MMP inhibition on long term potentiation. Hippocampal slices from aged rats were pretreated with a relatively dilute concentration of FN-439 (18 μM) for 4 hours prior to being introduced to the recording chamber. Once transferred to the recording chamber, slices were allowed a 1 hour wash-out phase prior to testing. Slices pretreated with MMP inhibitors experienced augmented LTP induction which persisted for at least 30min following tetanization (Figure 3A). This enhancement was also reflected in the total evoked dendritic depolarization during the recorded time (FIGURE 3B).

Example 5 (MMP inhibition promoted young-like fEPSP characteristics in slices from aged rats)

In addition to LTP deficits, slices derived from aged-rats also exhibited abnormalities in the temporal characteristics in the evoked field potentials. Specifically, aged field potentials reached maximum at a much quicker rate than younger animals, and failed to shift during potentiated conditions (see FIGURE 1). Applicants conceived that if these temporal features are associated with age related MMP elevations, they would likely be sensitive to MMP inhibition. In support of this conception, MMP inhibitor treated slices experienced a significantly greater peak-to-peak latency during baseline stimulatory conditions (i.e., a reduction in the temporal efficiency of the postsynaptic response) (two sample t-test; p<.0001) without a corresponding effect on amplitude (FIGURE 4A). This rightward shift represents a transformation of the fEPSP into a more young-like conformation. From this observation, applicants reasoned that the decreased temporal efficiency during baseline conditions would provide the latitude for a dynamic leftward shift (i.e., increased temporal efficiency) upon potentiation; a form of responsiveness which is more typical of young slices rather than old. Consistent with this conception, MMP inhibitor treated slices experienced a significantly greater decrease in peak-to-peak latency, for induction and early maintenance phases (FIGURE 4B), compared to non treated controls (two- factor ANOVA; p<.0001). In light of these results, and according to particular aspects of the present invention, age-associated MMP escalation is a source of burden on plasticity critical processes. Moreover, these data indicate that the detrimental effects of age-related MMPs increases on hippocampal function may be at least partially reversed bv MMP inhibition.

Example 6 (MMP inhibition failed to enhance LTP and alter fEPSP characteristics in slices from young rats)

To control for the unlikely possibility that beneficial effects of FN-439 pretreatment could be unrelated to MMP inhibition and due to some unknown, nonspecific effects on neuronal function, hippocampal slices generated from young animals were subjected to MMP inhibitor pretreatment and assessed for LTP and fEPSP characteristics. Due to the relatively low basal MMP levels present in hippocampal tissue applicants predicted that either the treatment would not have an effect on LTP and fEPSP characteristics or if the treatment did have an effect it would be detrimental to synaptic plasticity. Consistent with this prediction and applicants' present conception, the MMP inhibitor treatment conditions which produced LTP enhancement in aged slices produced slight deficits in LTP induction and early maintenance in young slices (FIGURES 5A, B). Furthermore, MMP inhibition did not affect peak-to-peak latency for baseline and post-tetanus conditions (FIGURES 5C- E). These findings further validate applicants' conception regarding the impact of elevated hippocampal MMP levels and further support applicants' interpretation and conception of how FN-439 administration affects hippocampal function in slices derived from aged rats.

Example 7

(Exogenous application of active MMPs impaired hippocampal LTP)

To further validate the conception that an increased MMP activity is antecedent to deficits to synaptic plasticity, applicants administered a 1-hour pre-iπcubation of an active MMP-3 fragment (bath concentration of 1nM) to a hippocampal slice preparation and monitored the effects on LTP. MMP-3- treated slices experienced a moderate reduction in LTP induction magnitude and increased maintenance decay were (FIGURE 6A). The impact of exogenous MMP-3 treatment on LTP induction and early maintenance was particularly reflected in total area of fEPSP, a measure of evoked dendritic depolarization, 30 minutes post tetanus (FIGURE 6B).

In addition to the effects on LTP, pretreatment with MMP-3 produced a leftward shift (i.e., decreased peak-to-peak latency) of the evoked field potentials without a corresponding effect on fEPSP amplitude (FIGURE 6C). Although post-tetanus modifications to peak-to-peak latency were unaffected the changes to plasticity and baseline peak-to-peak latency are coherent with the MMP-associated effects during aging.

Example 8

(The regulation of hippocampal MMPs by acute exposure to hydrogen peroxide was demonstrated) MMPs are induced by ROS under oxidative stress conditions for a variety of tissue types. However, this relationship between ROS and MMP modulation has not been reported in hippocampal tissue. To determine if

MMPs are regulated by ROS or modulated in experimental conditions used to induce oxidative stress in the hippocampus, applicants utilized a hippocampal slice preparation from 6-month-old animals. Hippocampal slices were treated with an acute (6 hours) exposure of the ROS hydrogen peroxide (H₂O₂) of concentrations typical for experimental oxidative stress test conditions. Upon

H₂O₂ treatment, we observed an increase in MMP-3/TIMP-2 complex formation (FIGURES 7A₁ B), but no change of total MMP-3 and MMP-9 protein levels . However, the fact that the H₂O₂ treatment failed to induce expressional changes for either MMP-3 or MMP-9 is not surprising considering that other studies reported ROS induction of MMPs required long term ROS exposure.

Example 9

(The cytoskeleton-regulatory protein cortactin was demonstrated to be regulated by MMP activity)

Plasticity within the hippocampal synaptic environment depends on subtle architectural remodeling as a result of activity-driven input. This architecture is constantly stabilized and destabilized by the interactions among a multitude of plasticity-associated molecules involved in modulating cell surface interactions, cell signaling and cytoskeletal stability. Previously, the present applicants found that the hippocampal levels of the actin cytoskeleton-regulatory molecule, cortactin, are regulated by MMP activity (FIGURE 8; also (Meighan, Meighan et al. 2006)). Specifically, during learning when MMP levels are elevated, hippocampal cortactin levels are low. Furthermore, if rats are administered MMP inhibitor during this time, cortactin levels are dramatically elevated indicating an inverse relationship between MMPs and cortactin. We now show that this inverse relationship between MMP activity and cortactin was observed in aged-hippocampus as well. In hippocampal tissue from aged rats, where MMP levels are dramatically elevated, cortactin levels are attenuated (FIGURE 9). Cortactin functions to stabilize the actin cytoskeleton and thus stabilizes dendritic spine structure. According to particular aspects of the present conceptions, it is likely that the excessive cortactin loss associated with chronic MMP elevation facilitates a destabilization of dendritic cytoskeletal elements and retraction of dendritic spines, leading to an overall loss of functional synapses. Consistent with this conception and relationship, a recent manuscript was published describing a disruption in F-actin and the loss of dendritic spines in hippocampal neurons as a direct result of MMP-7 upregulation (Bilousova, Rusakov et al. 2006). Therefore, it is likely that additional plasticity critical proteins are also affected or disrupted by heightened MMP activity.

Example 10

(Increased MMP levels were shown to be correlated with decreased weight of the aged hippocampus)

It is likely that hippocampal atrophy plays a significant role in the development of cognitive aging. Current evidence suggests that age- associated hippocampal atrophy is linked to dendritic field density reduction rather than cellular loss. Consistent with this, age associated increased MMP activity is concomitant to cortactin downregulation (see FIGURE 9), a situation likely to result in destabilization of structural elements reliant on actin filaments. Due to plausible connections between aging, MMP hyper- induction, cortactin downregulation and hippocampal atrophy, applicants conceived that increased MMPs are likely to be linked to neurodegeneration. To make an initial assessment, the mass of dissected hippocampi from 24- month old Sprague Dawley rats were measured and related to their corresponding active MMP-3 levels by regression analysis (FIGURE 10). The relationship between active MMP-3 and hippocampal mass was determined to be such that increasing presence of MMP-3 is associated with decreasing hippocampal mass. Significantly, this relationship is nonexistent in 3-month old control animals.

Example 11 (Effects of MMP inhibition on Morris water maze learning for aged-adult rats were demonstrated)

MMPs are known to be critical for normal neuronal functioning during learning in young adult rats. However, as demonstrated herein, excessive MMP levels may have serious consequences for hippocampal function. The present applicants have described herein that MMP inhibition improves the electrophysiological characteristics of the aged hippocampus, and further conceive that these inhibitors will also ameliorate age-associated cognitive decline. The primary goal of this Example was to confirm that MMP inhibitors are effectual at improving age-associated learning deficits in aged rats performing a spatial learning task, the Morris water maze. To validate this conceptive aspect, the exemplary MMP inhibitor FN439 was administered intraperitoneally to 22-month old F344 rats and their learning ability assessed in the water maze. Significantly, aged-adult rats treated with FN-439 were more efficient in the navigation of the water maze task than untreated rats (FIGURES 11A,B). Furthermore, relative to the untreated controls, FN-439- treated rats were more successful at locating a hidden pedestal within the first three days (TABLE 2). TABLE 2 is a contingency table describing the effect of MMP inhibition of aged-adult rats on successful completion of water maze task for days 1-3. Animals were allotted 120s, for each trial, to successfully complete water maze task. Successful completions (the ability to locate pedestal in allotted time) were scored as a "hit" and unsuccessful completions (inability to locate pedestal in allotted time) were scored as a "miss." A contingency table was constructed using pooled data from experiments where MMP inhibitor is administered ICV (n=3 animals) and IP (n=9 animals for treated and n=11 for control). Exact tests used to compare proportion of successful to unsuccessful maze completions between MMP treated and control rats (reported P-values are Holmes corrected for multiple comparisons). On average, the MMP inhibitor-treated rats were nearly 3-times as likely to locate the pedestal, during the first 3 days of training, than the untreated rats.

TABLE 2. Representative contingency table describing the effect of MMP inhibition of aged-adult rats on successful completion of water maze task for days 1-3

These data confirm that administration of the exemplary MMP inhibitor FN-439 two hours prior to water maze acquisition training improved the acquisition rate of the task in these animals. Significantly, this result is in contrast to behavioral data demonstrating inhibition of task acquisition for younger rats (Meighan, Meighan et al. 2006), and underscores the previously unanticipated aspect of applicants' novel and surprising conceptions as presently disclosed.

The present study provides the first evidence that a MMP-3 inhibitor can impair the acquisition of an associative memory task. This finding is important because it supports a causal relationship between learning-induced hippocampal MMP-3 activation and the formation of associative memories.

Furthermore, the results of the present Example highlight the unique importance of transient MMP-3 for learning. Significantly, therefore, in view of the herein disclosed negative correlation between cognitive ability and sustained markedly high levels of MMP-3 in aged rats, administration of specific or substantially specific MMP-3 inhibitors for treatment of age-related cognitive impairment (e.g., age-related cognitive decline) is a particularly preferred embodiment of the present invention. Additional embodiments provide methods comprising inhibition of MMP-3 and at least one other MMP

(e.g., MMP-9, etc.).

Example 12

(Application of MMP Inhibitors to Aged Animals (e.g., rats))

According to particular aspects of the present invention, MMP inhibitors may be administered to aged animals (e.g., for rats, intracerebroventricularly over a 14 day period using time-release formulations and/or osmotic minipumps). For rats, following a six-day pre-treatment period, the animals may be tested in the Morris Water Maze for eight consecutive days. In particular embodiments, one or more specific and/or broad-spectrum MMP inhibitors may be employed. For example, a first group of rats may be given a FN-439-based formulation (e.g., Calbiochem, Cat. No. 444250), which is a hydroxymate-based MMP inhibitor. According to preferred aspects of the present invention, one or more

MMP inhibitors is administered to an animal in need thereof in an amount that is therapeutically effective in treating age-related cognitive impairment. In preferred aspects an inhibitor of MMP-3 and/or MMP-9 is used for this purpose.

Example 13

(Use of MMP Inhibitors to Treat Cognitive Decline in Humans) According to additional aspects of the present invention, MMP inhibitors (e.g., as disclosed herein) may be administered to aged humans to treat age-related cognitive decline. In particular embodiments, one or more specific and/or broad-spectrum MMP inhibitors (e.g., as disclosed herein) may be employed for this purpose. In alternate embodiments, MMP-specific inhibitors are used (e.g., MMP-3 and/or MMP-9 inhibitors). According to preferred aspects, one or more MMP inhibitors is administered to human subject in need thereof in an amount that is therapeutically effective in treating age-related cognitive impairment. In preferred aspects an inhibitor of MMP-3 and/or MMP-9 is used for this purpose. In preferred embodiments, at least MMP-3 is inhibited. In alternate preferred embodiments, MMP-3 is inhibited along with at least one other MMP (e.g., MMP-9, etc.).

The preceding Examples are included for illustrative purposes only, and are not intended to limit the scope of the presently claimed subject matter.

Claims

1. A method for treating cognitive impairment, comprising administration to a subject in need thereof a therapeutically effective amount of at least one matrix metalloproteinase (MMP) inhibitor sufficient to provide for at least one of precluding, alleviating, reversing, or inhibiting cognitive impairment characterized by persistent or sustained elevated expression and/or activity of at least one MMP.

2. The method of claim 1, wherein the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least one MMP having the characteristic elevated expression or activity.

3. The method of claim 1, wherein the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least two MMPs having the characteristic elevated expression or activity.

4. The method of claim 1, wherein the at least one matrix metalloproteinase (MMP) inhibitor is MMP-specific or substantially specific to a particular MMP or to a limited number of MMPs from about one to about four MMPs.

5. The method of claim 1, wherein the cognitive impairment is characterized by sustained elevated MMP-3 expression and/or activity, and wherein the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit at least matrix metalloproteinase 3 (MMP-3).

6. The method of claim 5, wherein the cognitive impairment is further characterized by sustained elevated expression and/or activity of at least one additional MMP, and wherein the at least one matrix metalloproteinase (MMP) inhibitor is suitable to inhibit the additional matrix metalloproteinase (MMP).

7. The method of claim 6, wherein the at least one additional matrix metalloproteinase (MMP) comprises MMP-9.

8. The method of claim 1, wherein the at least one metalloproteinase (MMP) inhibitor comprises a hydroxamate moiety.

9. The method of claim 1, wherein the at least one metalloproteinase (MMP) inhibitor comprises a zinc-binding hydroxamate moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase.

10. The method of claim 1, wherein the at least one metalloproteinase (MMP) inhibitor comprises a non-hydroxamate zinc-binding moiety and a peptide or peptidomimetic backbone capable of binding at least one matrix metalloproteinase.

11. The method of claim 1, wherein treating comprises prophylactic administration.

12. The method of claim 1 , wherein treating comprises administration to alleviate cognitive impairment.

13. The method of claim 1, wherein treating comprises administration to inhibit cognitive impairment.

14. The method of claim 1 , wherein treating comprises administration to reverse cognitive impairment.

15. The method of claim 1, wherein the cognitive impairment characterized by sustained elevated MMP expression and/or activity is age- related.

16. The method of claim 1, wherein the subject is determined to have age-related cognitive impairment.

17. The method of claim 7, wherein the subject is determined to have age-related cognitive decline, dementia or Alzheimer's disease.

18. The method of claim 1, further comprising combination therapy, wherein the at least one matrix metalloproteinase (MMP) inhibitor is used in conjunction with at least one additional therapeutic agent.

19. The method of claim 18, wherein the at least one additional therapeutic agent is selected from the group consisting of: standard non- steroidal anti-inflammatory drugs (NSAID'S), piroxicam, diclofenac; a propionic acid, naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen; a fenamate, mefenamic acid, indomethacin, sulindac, apazone; a pyrazolone, phenylbutazone; a salicylate, aspirin; an analgesic or intraarticular therapy, a corticosteroid; a hyaluronic acid, hyalgan, synvisc; an immune suppressant, cyclosporine, interferon; a TNF-.alpha. inhibitor, Enbrel™; low dose methotrexate, lefunimide, hydroxychloroquine, d-penicilamine, auranofin, parenteral gold and oral gold.

20. The method of claim 18, wherein the at least one additional therapeutic agent is selected from the CNS agent group consisting of: an antidepressant, sertraline, fluoxetine, paroxetine; an anti-Parkinsonian drug; deprenyl, L-dopa, requip, miratex; a MAOB inhibitor, selegine, rasagiline; a COMP inhibitor, tolcapone, Tasmar; an A-2 inhibitor, a dopamine reuptake inhibitor, an NMDA antagonist, a nicotine agonist, a dopamine agonist, an inhibitor of neuronal nitric oxide synthase, an anti-Alzheimer's drug; an acetylcholinesterase inhibitor, metrifonate, donepezil, Aricept, Exelon, ENA 713 or rivastigmine; tetrahydroaminoacridine, Tacrine, Cognex, or THA; a COX-1 or COX-2 inhibitor, celecoxib, Celebrex, rofecoxib, Vioxx; propentofylline, an anti-stroke medication, an NR2B selective antagonist, a glycine site antagonist, and a neutrophil inhibitory factor (NIF).

21. The method of claim 18, wherein the at least one additional therapeutic agent is selected from the group consisting of: an estrogen; a selective estrogen modulator, estrogen, raloxifene, tamoxifene, droloxifene, lasofoxifene; an agent that results in reduction of A.beta.1-40/1 -42, an amyloid aggregation inhibitor, a secretase inhibitor; an osteoporosis agent, droloxifene, fosomax; immunosuppressant agents, FK-506, rapamycin; an anticancer agent, endostatin, angiostatin; a cytotoxic drug, adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere; an alkaloid, vincristine; an antimetabolite, methotrexate; a cardiovascular agent, calcium channel blockers; a lipid lowering agent, a statin; a fibrate, a beta-blocker, an ACE inhibitor, an angiotensin-2 receptor antagonist , and a platelet aggregation inhibitor.

22. The method of claim 1 , wherein the at least one matrix metalloproteinase (MMP) inhibitor is at least one selected from the group of compounds of Table 1 disclosed herein.

23. The method of claim 8, wherein the at least one matrix metalloproteinase (MMP) inhibitor comprises 4-Abz-Gly-Pro-D-Leu-D-Ala-NH- OH or a derivative thereof, wherein Abz is aminobenzoyl.