US20100028839A1

US20100028839A1 - Methods and systems for evaluating memory agents

Info

Publication number: US20100028839A1
Application number: US12/505,454
Authority: US
Inventors: Timothy P. Tully; Lila Davachi
Original assignee: Dart Neuroscience LLC
Current assignee: Dart Neuroscience Cayman Ltd
Priority date: 2008-07-18
Filing date: 2009-07-17
Publication date: 2010-02-04
Also published as: US8462179B2; US20100013857A1

Abstract

The present invention provides methods and systems for identifying, evaluating, and testing various compounds as memory agents and regimens as training protocols related to memory enhancement and/or impairment. Such methods and systems may test any compounds or protocols, including the memory agents and training protocols described herein. The methods can comprise combining training protocols with the general administration of memory agents. In some embodiments, the present invention may involve identifying, selecting, testing, evaluating or assessing a compound as a drug candidate for a memory agent, for example, as one effective for enhancing or impairing memory.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application Ser. No. 61/081,845, filed Jul. 18, 2008, the entire content of which is fully incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to methods and systems of evaluating, identifying or assessing the effectiveness of memory agent and/or training protocols administered to subjects.

BACKGROUND OF THE INVENTION

Memory is the recollection of past experiences. One is able to register new information, store it and then recall it sometime later. Recollection of facts and events represents a form of conscious, or explicit, memory. One also remembers less conscious skills, such as riding a bike, playing a musical instrument or associating simple cues with reward or punishment, which represent more implicit forms of memory. For both of these cases, the process of memory formation appears to proceed through three general stages. Acquisition (learning) involves the initial perception of a new experience. Short-term memory of this newly acquired experience then appears transient and labile. Under appropriate conditions, often requiring repetition and rest, short-term memory of an experience then can be “consolidated” into a more durable long-term memory.
An every-day example of this memory process involves using a phone number. When one first looks up a new phone number, they recite it to themselves a few times to learn it (learning). One then can dial the number successfully within seconds to minutes, if not distracted. Interruptions, on the other hand, almost always cause one to forget the number. Minutes to hours after dialing the number, one most likely would not remember the number anymore (short-term memory). If the phone number corresponds to that of a friend's new cell phone, however, one might find themselves dialing it repeatedly every day or so. With enough repetition, one eventually can recall the phone number through long-term memory.
The human brain is evolutionarily designed to sense its internal and external environment, to perceive causal relations among stimuli and to change its response adaptively. To accomplish this incredible task, a self-regulating network of 40 thousand genes directs the development of a cellular network of one hundred billion neurons with 100 trillion connections among them. Such complexity is staggering. Yet neurobiologists have begun to understand how the human brain works in part by studying brain function in other animals.
New experiences obviously can be quite complex, but when reduced in the laboratory to simple, controllable studies of “associations” between two identified stimuli (Pavlovian learning, for instance), the process of memory formation can be studied in many different animals ranging from insects to mammals. These animal studies have contributed to our current understanding of how memory formation proceeds, revealing aspects of neuroanatomy and biochemistry of the brain. Remarkably, these studies have established that much of the basic biochemistry and behavioral phenomenology of memory formation has been evolutionarily conserved throughout the animal kingdom, even though neuroanatomy differs appreciably among species.
These animal studies have shown that memory formation generally proceeds through several functionally distinct temporal phases. Acquisition (LRN) usually requires repetition and is thought to recruit maximal participation of the underlying neural circuitry that normally registers such an experience. Once learning occurs, early memory tends to decay rapidly and is sensitive to disruption by distraction and anesthesia (i.e., anesthesia-sensitive memory; ASM). Genetic dissection of ASM in fruit flies has shown that ASM can be further decomposed into short-term memory (STM) and intermediate-term memory (ITM).
By contrast, long-lasting memory becomes resistant to disruption (i.e., anesthesia-resistant memory; ARM) and persists for long periods. Importantly, these animal studies generally have revealed that protein synthesis is required for the appearance of long-term memory (LTM). Once again, fruit fly studies have revealed that (i) LTM, but not ARM, requires protein synthesis and (ii) ARM and LTM can be genetically dissected into functionally independent phases of memory.
Animal studies also have revealed that the basic wiring diagram of the brain emerges during development. Most connections (synapses) among neurons, however, are crude and non-functional. Subsequent experience is required to refine these synaptic connections, thereby fine tuning the functions of various circuits. Activity-dependent synaptic reorganization continues within the adult brain, as well. Learning a new experience initiates a neural activity, which ultimately produces synaptic reorganization among neurons in the relevant circuit. These synaptic changes (synaptic plasticity), distributed throughout the neural circuit, are considered to be the physical basis of long-term memory.
Experience-dependent changes in neural activity initiate within neurons a complex biochemical response. The ultimate effects of this biochemistry are to produce short-term (transient) and long-term (long-lasting) synaptic plasticity. Basic research continues to identify more and more of the signaling pathways and other cell biology that underlie this synaptic plasticity. The first of these to be discovered was the cAMP pathway, a cascade of enzymatic reactions that transduces extracellular neurotransmitter release into intracellular changes in the functional states of many proteins. Neurotransmitters, their corresponding receptors, and cytoplasmic PKA appear to regulate short-term plasticity, while activation of the transcription factor CREB (cAMP response element binding protein) is required to produce long-term plasticity.
In all species studied so far, the CREB gene is expressed in two opposing forms—an activator or a repressor of downstream, CRE-mediated gene transcription. Key behavioral experiments in Drosophila originally established a critical role for CREB in the formation of long-term memory. Normal fruit flies are capable of learning the association between a particular smell and the punishment of foot shock. After one training session, a majority of flies will avoid the previously shock-paired odor when given a choice between it and a neutral odor in a T-maze. Though such learning initially is quite robust, memory thereafter is transient, decaying away within a day. In contrast, a bona fide protein synthesis-dependent LTM lasting more than a week can be produced in normal flies when subjected to ten training sessions with a 15-min rest interval between them. In flies genetically engineered to produce high levels of CREB repressor, LTM specifically is blocked with no effects on learning or early memory. In flies genetically engineered to produce high levels of CREB activator, LTM after spaced training is not affected but, surprisingly, is induced after just one training session. In short, overexpression of CREB activator enhances memory by reducing the number of training sessions required to induce the formation of LTM—the functional equivalent of a photographic memory. Molecular studies in several animal models have begun to elucidate further the biochemistry of plasticity and to link various aspects of synaptic plasticity with memory formation, leading to the following general view of brain plasticity: a new experience is registered in a specific neural circuit as a change in neural activity. This neural activity induces a complex inter- and intra-cellular biochemical response that still is being discovered. To date, however, a few key aspects of the process appear evolutionarily conserved. Stimulation of the NMDA receptor appears central to induction of both the local, transient biochemical response at the synapse and the long-lasting biochemical response in the cell nucleus (phosphorylation of CREB). Several intracellular signaling pathways appear to be induced initially by neural activity, but CREB nonetheless appears to be a dominant “downstream” target, regulating the conversion of early memory to lasting memory. Down-regulation of CREB suppresses, while up-regulation of CREB enhances, a gene transcriptional response underlying a synaptic grow process that yields lasting functional and structural plasticity at synapses. In essence, CREB appears to act as a master switch that determines when neural activity will give rise to lasting, structural changes in a circuit. A key factor influencing this switch is repetition; when the CREB pathway is up-regulated, the number of training sessions required for the formation of long-term memory is decreased.
Four basic objectives guided the approach to screen for drugs that modulated the “CREB pathway.” First, for efficiency, the screen had to be high throughput, amenable to robotic manipulations in a 96-well plate format. Second, because any identified drug was ultimately to be used in humans, the preferable target was an endogenous (neuronal) form of human CREB. Third, when designing the screen, all the relevant upstream signaling pathways that might impinge on CREB function during memory formation were not yet known. Therefore, a broad-based screen, monitoring CREB-dependent gene expression as a functional readout of the CREB pathway seemed more inclusive of any missing, yet potentially important, biochemical signaling. Finally, the screen needed to identify drugs that had no effect on CREB function alone but rather synergized with co-activation of cAMP signaling. This requirement was designed to mimic the original behavioral experiments. In flies, overexpression of CREB alone had no effect on fly behavior. Rather, training per se was required to produce enhanced memory, because experience-induced increases in neural activity activated the cAMP pathway.
These considerations were incorporated into a high throughput screen (HTS) of cultured human neuroblastoma cells, which were stably transfected with a CRE-luciferase reporter gene. In this manner, changes in CREB-dependent transcription of luciferase could be monitored via fluorescence. To mimic (at the biochemical level) the associative requirement for memory formation, drugs were sought that produced no change in luciferase activity on their own but synergized (>2-fold) with a suboptimal dose of forskolin, which activates the cAMP pathway (as does neural activity in vivo). Using a robotic system to screen these cells in 96-well plates, large libraries of drug compounds have been screened efficiently, yielding numerous “active hits.” To confirm, these active hits then were assessed at four different concentrations with or without forskolin. Several Confirmed Actives emerged from this screening process.
Interestingly, an efficacious molecular target was discovered to be phosphodiesterase-4 (PDE4). Generally and specifically, this discovery makes sense. Most biochemical pathways function within a well-regulated optimum, with some molecular components accelerating (positive regulators), and others retarding (negative regulators), signal propagation. Given the opposing functions of CREB activator and repressor isoforms, this notion applies to regulation of the CREB switch during long-term memory formation. Like genetic mutations, most drug compounds actually serve as inhibitors of target function (“antagonists” for drugs and “hypomorphs” for mutations). Hence, discovering drug compounds that antagonize negative regulators of a pathway might be expected often from a screen for enhanced function. This, of course, is the case for a PDE inhibitor; decreasing PDE activity leads to an increase of cAMP normally synthesized by adenylyl cyclase (AC), and elevated cAMP leads to activation of CREB via PKA. Moreover, a PDE inhibitor might be expected to have minimal effect alone, in the absence of forskolin-induced activation of AC (or neural activity-induced activation of AC in vivo).
An advanced PDE-4 inhibitor, HT-0712, has been shown to be a validated compound for clinical development, along with several other CREB pathway enhancers in addition to HT-0712. To date, behavioral assessments in rodent models of memory formation and biochemical experiments have shown that these compounds effectively enhance memory.
It is important to develop, identify and evaluate such compounds and therapies. Such therapies may include particular training protocols and/or administration of such compound or compounds, including compounds that serve as memory agents, which may be memory enhancers that specifically up-regulate the CREB pathway, as well as memory impairers.

SUMMARY OF THE INVENTION

The present invention includes methods including the steps of assessing the effectiveness of a memory agent administered to a subject, wherein said assessing comprises presenting to the subject sets of stimuli and evaluating the subject's response to the stimuli. The assessing may include a first period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a criterion; and a second period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a long term success measurement. In some embodiments, the second period begins at least about one day after the completion of the first period. The evaluating of the subject's response may include comparing the criterion to the long term success measurement. The sets of stimuli may include a plurality of pairs of stimuli. In some embodiments, the each of the plurality of pairs of stimuli can include a positive element and/or a negative element. In other embodiments, each of the plurality of pairs of stimuli may include an element that has an identifiable association with the other element of the pair. In some embodiments, this identifiable association may be a face—name association or word—word association. The subject may be an animal. In certain embodiments, the animal may be a primate, a mammal, a mouse or a rat. In some embodiments, the primate may be a human, a monkey, a lemur, a macaque or an ape. In some embodiments, the first period may be from about one to about ten days. In some embodiments, the second period may be about one day. In some embodiments, there may be a plurality of subjects, which may include a control group and an experimental group. In certain embodiments, the assessing may include comparing the control group to the experimental group. In some embodiments, the evaluating of the subject's response includes calculating and/or measuring one or more aspects selected from the group consisting of: the ratio of the long term success measurement to the criterion, long term memory retention, the amount of time required to achieve a particular criterion, and/or the amount of time between the first period and the second period while keeping a constant, near-constant, or improved ratio of the long term success measurement to the criterion.
In other embodiments, the present invention may involve a method including selecting a memory agent as a drug candidate, wherein said selecting includes the steps of: administering a memory agent to a subject; and presenting to the subject sets of stimuli and evaluating the subject's response to the stimuli. The assessing may further include: a first period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a criterion; and a second period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a long term success measurement. In some embodiments, the second period begins at least about one day after the completion of the first period. In certain embodiments, the evaluating of the subject's response comprises calculating and/or measuring one or more aspects selected from the group consisting of: the ratio of the long term success measurement to the criterion, long term memory retention, the amount of time required to achieve a particular criterion, and/or the amount of time between the first period and the second period while keeping a constant, near-constant, or improved ratio of the long term success measurement to the criterion. In some embodiments, the sets of stimuli comprise a plurality of pairs of stimuli. In some embodiments, the subject may be an animal, including an mammal such as a primate, a mouse and a rat.
In yet other embodiments, the present invention involves a method including: testing a memory agent as a long term memory enhancer, wherein said testing includes the steps of: administering a memory agent to a subject; and presenting to the subject sets of stimuli and evaluating the subject's response to the stimuli. In certain embodiments, the assessing further includes: a first period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a criterion; and a second period comprising presenting to the subject stimuli associated into sets and evaluating the subject's response to the stimuli to establish a long term success measurement. In some embodiments, the second period begins at least about one day after the completion of the first period. In some embodiments, the evaluating of the subject's response comprises calculating and/or measuring one or more aspects selected from the group consisting of: the ratio of the long term success measurement to the criterion, long term memory retention, the amount of time required to achieve a particular criterion, and/or the amount of time between the first period and the second period while keeping a constant, near-constant, or improved ratio of the long term success measurement to the criterion. In certain embodiments, the sets of stimuli comprise a plurality of pairs of stimuli. In some embodiments, the subject may be an animal, including an animal selected from the group consisting of a primate, a mammal, a mouse and a rat.
In yet further embodiments, the present invention may be a method including: assessing the effectiveness of a memory agent, wherein said assessing comprises the steps of: providing a first subject and a second subject, wherein the first subject and the second subject are of the same species; administering a memory agent to the first subject; presenting the first subject and the second subject a plurality of pairs of stimuli during a first period and evaluating the first subject and the second subject's responses to the stimuli to establish a criterion for each of the first subject and the second subject; presenting the first subject and the second subject the plurality of pairs of stimuli during a second period and evaluating the first subject and the second subject's responses to the stimuli to establish a long term success measurement for each of the first subject and the second subject; comparing the first subject and the second subject's criterions to their respective long term success measurements. In some embodiments, the second period begins at least about one day after the completion of the first period. In some embodiments, the second period begins from about 1 to about 10 days following the completion of the first period. In certain embodiments, the comparing of the criterions to the long term success measurements comprises: establishing a first ratio of the first subject's long term success measurement to the first subject's criterion; establishing a second ratio of the second subject's long term success measurement to the second subject's criterion; and comparing the first ratio to the second ratio. In certain embodiments, the sets of stimuli include a plurality of pairs of stimuli. In some embodiments, the plurality of pairs of stimuli comprises a positive element and/or a negative element. In other embodiments, each of the plurality of pairs of stimuli comprises an element that has an identifiable association with the other element. In some embodiments, the identifiable association is selected from the group consisting of a face—name association and a word—word association. In some embodiments, each of the first subject and the second subject is an animal, including an animal is selected from the group consisting of a primate (including a human, a monkey, a lemur, a macaque and an ape), a mammal, a mouse and a rat. In certain embodiments, the first period comprises from about one to about ten days. In some embodiments, the second period comprises about one day. In some embodiments, each of the first subject and the second subject is a plurality of subjects.
In some embodiments, the present invention includes methods similar to those recited above, but which focus on evaluating an training protocol rather than or in addition to a memory agent. Some embodiments involve a method including the steps of: assessing the effectiveness of an training protocol administered to a subject, wherein said assessing comprises presenting to the subject sets of stimuli and evaluating the subject's response to the stimuli. Other embodiments involve a method including: testing a training protocol as a long term memory enhancer, wherein said testing includes the steps of: administering an training protocol to a subject; and presenting to the subject sets of stimuli and evaluating the subject's response to the stimuli. Further embodiments involve a method including: assessing the effectiveness of an training protocol, wherein said assessing includes the steps of: providing a first subject and a second subject, wherein the first subject and the second subject are of the same species; administering an training protocol to the first subject; presenting the first subject and the second subject a plurality of pairs of stimuli during a first period and evaluating their responses to the stimuli to establish a criterion for each of the first subject and the second subject; presenting the first subject and the second subject the plurality of pairs of stimuli during a second period and evaluating their responses to the stimuli to establish a long term success measurement for each of the first subject and the second subject; comparing the criterions of the first subject and the second subject to their respective long term success measurements.
The present invention also includes systems, including systems for use along with the methods disclosed herein. For example, in some embodiments, the system may include all elements necessary to perform an embodiment of the methods of the present invention. Systems of the present invention may be useful for selecting a memory agent as a drug candidate, assessing the effectiveness of a memory agent, assessing the effectiveness of a training protocol, and/or testing a memory agent as a long term memory enhancer, and/or testing a memory agent along with an training protocol as a long term memory enhancer.
In some embodiments, a system of the present invention includes sets of stimuli, wherein said sets of stimuli are configured to permit evaluation of a subject's response to the sets of stimuli and thereby assess the effectiveness of an training protocol administered to the subject. In some embodiments, the sets of stimuli comprise a plurality of pairs of stimuli. In certain embodiments, each of the plurality of pairs of stimuli comprises a positive element and/or a negative element. In some embodiments, each of the plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of the pair. In further embodiments, the identifiable association is selected from the group consisting of a face—name association and a word—word association. In other embodiments, the system may further include testing protocols, testing schedules, and/or memory agent dosage and administration schedules.
In other embodiments a system of the present invention includes sets of stimuli, wherein said sets of stimuli are configured to permit evaluation of a subject's response to the sets of stimuli and thereby assess the effectiveness of a memory agent administered to the subject. In some embodiments, the sets of stimuli comprise a plurality of pairs of stimuli. In various embodiments, each of the plurality of pairs of stimuli comprises a positive element and/or a negative element. In other embodiments, each of the plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of the pair. In certain embodiments, the identifiable association is selected from the group consisting of a face—name association and a word—word association. In other embodiments, the system may further include testing protocols, testing schedules, and/or memory agent dosage and administration schedules.
In some embodiments, the methods and systems of the present invention utilize a memory agent that is one or more selected from the group consisting of a compound having the structure:
wherein each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl or C₅-C₇cycloalkenyl; —F, —Cl, —Br, or —I; —NO₂; —N₃; —CN; —OR₄, —SR₄, —OCOR₄, —COR₄, —NCOR₄, —N(R₄)₂, —CON(R₄)₂, or —COOR₄; aryl or heteroaryl; or any two of Y₁, Y₂, Y₃and Y₄present on adjacent carbon atoms can constitute a methylenedioxy group; wherein each R₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl, C₅-C₇cycloalkenyl, aryl or aryl(C₁-C₆)alkyl; wherein A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; wherein A′ is
wherein R₁and R₂are each independently H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, or —CN; wherein R₃is H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, —CN, —OR₆, aryl or heteroaryl; wherein R₅is straight chained or branched C₁-C₇alkyl, —N(R₄)₂, —OR₄or aryl; wherein R₆is straight chained or branched C₁-C₇alkyl or aryl; wherein B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-napthyridinyl, pteridinyl, or phthalimidyl; provided however, that the carbon atom or carbon atoms ortho to the nitrogen atom of the imine bond may only be substituted with one or more of the following —F, —Cl, —Br, —I, —CN, methyl, ethyl or methoxy; wherein n is an integer from 1 to 4 inclusive; wherein aryl is phenyl or napthyl, including phenyl and napthyl substituted with one or more of the following: —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkynyl, C₃-C₇cycloalkyl, C₁-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —OR₄, SR₄, —OCOR₄, —COR₄, —NCOR₄, —CO₂R₄, —CON(R₄)₂or (CH₂)_n—O—(CH₂)_mCH₃; or a pharmaceutically acceptable salt thereof.
In some embodiments, the methods and systems of the present invention utilize a memory agent that is one or more selected from the group consisting of a compound having the structure:
wherein Me is methyl, cPent is cyclopentyl and the 3 and 5 carbons are in the S configuration.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B illustrate graphs showing results from an embodiment of the present invention described in Example 1. Results are expressed as mean days±s.e.m per treatment, *p<0.05 versus vehicle.

FIGS. 2A-2B illustrate graphs showing results from an embodiment of the present invention described in Example 1. Results are expressed as mean percent performance±s.e.m. per treatment.

FIGS. 3A-3B illustrate graphs showing results from an embodiment of the present invention described in Example 1. Results are expressed as mean percent performance±s.e.m. per treatment.

FIGS. 4A-4D illustrate exemplary pairs of visual stimuli that may be used in an embodiment of the present invention described in Example 1.

FIG. 5 illustrates a graph showing performance of healthy elderly volunteers in a paired-associate (face-name) memory assay embodiment described in Example 2. Subjects were asked to memorize a set of 40 pairs of faces and names (professions) for 6 days (d2-d7). On training days, subjects went through the list of 40 pairs only once. They were queried for their recall first and then given the correct answers to continue training. After training, three-day memory retention was quantified using only face-name pairs 1-20 (d10). Seven-day memory retention was quantified again for these face-name pairs 1-20 (d14b). Seven-day retention also was quantified for face-name pairs 21-40 twice (d14a and d14a2), with a 1-min interval between recall sessions.

DETAILED DESCRIPTION

In the following paragraphs, the present invention will be described by way of example with reference to the attached figures. Throughout this description, the preferred embodiments and examples shown should be considered as exemplars, rather than as limitations on the present invention. As used herein, the “present invention” refers to any one of the embodiments of the invention described herein, and any equivalents. Furthermore, reference to various feature(s) of the “present invention” throughout this document does not mean that all claimed embodiments or methods must include the referenced feature(s).
The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more compounds and includes equivalents thereof known to those of ordinary skill in the art and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
The present invention provides methods and systems for identifying, evaluating, and testing various compounds as memory agents and regimens as training protocols. Such methods and systems may test any compounds or protocols, including the memory agents and training protocols described herein. The methods can comprise combining training protocols with the general administration of memory agents. In some embodiments, the present invention may involve identifying, selecting, testing, evaluating or assessing a compound as a drug candidate for a memory agent, for example, as one effective for enhancing memory.
A drug candidate may be a compound that has been identified through a drug discovery process and has been synthesized, characterized, optimized, and/or screened or assayed. Once a compound has been shown to have potential for a given activity or effect, for example by use of the methods or systems of the present invention, it may be a drug candidate—i.e., one that may be put through clinical trials with the intent that it be ultimately commercialized.

Methods of Identifying, Evaluating and Testing Memory Agents and Training Protocols

In some embodiments, the present invention involves the use of certain tasks to assess one or more aspects of an animal's memory capabilities. For example, the animal's learning capabilities, short term memory, or long term memory retention may be tested and/or evaluated. As used herein, the term “memory,” without an additional modifier (e.g., short term memory), refers to long term memory. As used herein, the term “memory agent” refers to memory augmenting agents or memory suppressing agents. Memory augmenting and suppressing agents include, but are not limited to, those molecules, compounds, and/or agents that act on a CREB pathway, as described, for example, in U.S. Pat. Nos. 5,929,223, 6,051,559, 6,689,557, and 6,890,516, and in U.S. patent application Ser. No. 11/066,125, the disclosure of each of which is incorporated by reference in their entirety herein.
As used herein, the term “animal” includes mammals, as well as other animals, vertebrate, invertebrate (e.g., birds, fish, reptiles, insects (e.g., Drosophila species) and mollusks (e.g., Aplysia)). The terms “mammal” and “mammalian”, as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include, without limitation, humans and primates (e.g., monkeys, chimpanzees, humans, apes, lemurs, or macaques), rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses).
The animal can be an animal with some form and degree of memory dysfunction or an animal with normal memory performance (i.e., an animal without any form of memory failure (dysfunction or loss of any memory)). One or more animals may be used and such animals may be placed in one or more groups (e.g., one or more control groups and/or one or more experimental groups). The animal may be any suitable age.
The tasks may involve the presentation of certain stimuli to the subjects. The stimuli may be any suitable sensory stimulus including, without limitation, auditory, visual, tactile, olfactory, gustatory and/or combinations thereof. In some embodiments, the stimuli may be visual and be one or more of a symbol, letter, number, line, shape, color, picture or combinations thereof. The stimuli may be associated into sets, such that a certain relationship is established between the members of the sets. The stimuli may be associated into groups of about 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some such embodiments, the stimuli are associated into pairs. In some embodiments, each stimulus in the pair is of the same type (i.e., audio, visual or tactile) while in other embodiments the pair may be of different types. In some embodiments, an element in the pair will have some identifiable association with the other element of the pair. An identifiable association can be a visual, audible, logical or other aspect of the one or more elements of the pair that would cause one to associate that element of the pair with its corresponding element. In other embodiments, there may be no identifiable association. For example, the pair could be a word-word pair wherein one word does not have an identifiable association with the other word (e.g., comb-apple). An example of visual paired stimuli is depicted in FIGS. 4A-4D. Another example of a stimulus pair with an identifiable association could be a face—name pairing and/or an occupation—face/name pairing. In some embodiments, there may also be an occupation listed along with the face-name pairing.
In some embodiments, the subjects may be presented one or more pairs of stimuli. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, 120, 150, or any other suitable number of pairs of stimuli may be presented to the subject. In some embodiments, one element of the pair is designated to be positive—meaning that the subject will receive a reward if it selects that element. In other embodiments, one element of the pair is designated to be negative—meaning that the subject will receive negative feedback if it selects that element. In some embodiments, the subject selects the element by touching the element, but in other embodiments any other suitable selection method may be used. In some embodiments, the reward may be food, but in other embodiments the reward may be a pleasant sound, access to certain toys or items, or anything suitable for triggering a positive response from the subject. In some embodiments, the negative feedback is a shock, a sound, a punishment or withholding of privileges, or any suitable action or the like that provides the subject with negative feedback.
The type of feedback, the selection of the pairs of stimuli, sets of pairs of stimuli, species and age of the subjects, and other aspects of the tasks may be modified to test and/or evaluate different aspects of a subject's memory. For example, using stimulus pairs wherein each element has an identifiable association to the other element may be used to evaluate the subject's relational memory.
In other embodiments, other evaluation methods other than the reward and/or negative feedback evaluation methods may be used. For example, in some embodiments, such as those where the subject is human, the stimuli may have a relation such that the subject may determine if the pairing or other association is correct or incorrect (e.g., are the stimuli paired such that the intended identifiable association between one element of a pair and the other element in the pair is present). For example, if a face—name pairing has been taught to the subject, the subject may be presented pairs of stimuli that have been scrambled such that the correct face—name pairing has been disrupted. The subject could then communicate whether the face—name pairing presented to it is the correct face—name pairing it has been taught. In such embodiments, no discrete reward would be necessary, or the reward could simply be feedback on the correctness of the human's responses. The pairs of stimuli may be presented in any suitable fashion. For example, the pairs may be presented in random, organized, or a pseudo-random order during each testing session.
The subjects may be tested over any suitable period at any suitable frequency. For example, the subjects may be tested 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, 365, or any other suitable number of times per testing event (e.g., if testing is daily, the subject may be tested once, twice, three times, etc). In such testing, subjects may be shown any suitable number of stimuli or pairs of stimuli. Subjects may have tests daily, every other day, weekly, bi-weekly, monthly, or any other suitable frequency. Moreover any suitable number of subjects may be tested. In some embodiments, testing is done in an effort to teach or encode the subject with the information as to the relationship between the paired stimuli. For example, the testing may be done to teach the subject which of the elements of the pair is the positive and/or negative element. In other embodiments, the testing may be done to teach the subject the correct pairing of elements within a set of stimuli (e.g., the correct face—name, occupation-name, or occupation-face/name association). In such embodiments, a plurality of phases of testing would likely be required. A first teaching phase, followed by a second phase wherein the subject's knowledge is tested by asking, through an appropriate method depending on the subject type (e.g., human vs. rat), if the presented pairings are correct.
The subjects may be evaluated in any suitable manner. In some embodiments, the subjects are presented with one or more pairs of stimuli in which one element of the pair is designated positive and/or one element of the pair is designated negative. In such an embodiment, the subject, having learned which element of the pair is the positive and/or negative element, is evaluated on the number of times it selects the positive and/or negative element and/or does not select the positive and/or negative element. In other embodiments, the pairs of stimuli may have a relation to one another (e.g., a face—name relation, occupation-name relation, occupation-face/name relation, a pair of words, a set of letters or numbers, parts of a picture or symbol). In such embodiments, the subject may be evaluated on its ability, having learned the correct relation between the elements of the pair, to re-establish the appropriate pairs (e.g., placing the name element with the corresponding picture element, or the like). In some embodiments, the pairs may be altered in some manner and the subject could be asked to identify the manner in which the pair was altered (for example, the response could be “intact,” “rearranged,” “one new,” or “both new”).
In some embodiments, a subject's long-term memory may be tested. In such embodiments, a criterion result can be established. Such a criterion may be established in a variety of manners. For example, in embodiments where a pair of stimuli is presented to the subject wherein one element of the pair is positive and/or one element of the pair is negative, the subject may be presented with the pair (or sets of pairs) until the subject identifies the positive and/or negative element with a certain frequency. That frequency would then serve as a criterion against which later results may be evaluated. Similarly, the frequency of the correct establishment of the relation between elements of a pair of stimuli may also be used to establish a criterion. In some embodiments, a subject is evaluated until it reaches a certain success rate. For example, a criterion of about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any other suitable percentage or number of correct results could be used as a target. In other embodiments, a subject may be tested a certain number of times or over a certain period of time, and the last evaluation may be used to establish the criterion. For example, a period of about 1-23 hours or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, or 365 days, or any other suitable number of hours or days may be used to establish the criterion. Also, the subject may be tested about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, 365, or any other suitable number of times to establish a criterion. The criterion may also be established using a mean, median, or other statistical measure of the subject's actual results.
In some embodiments, once the criterion is established, the subject is not evaluated or shown any of the pairs of stimuli used to establish the criterion for a certain amount of time. This amount of time may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, 365, or any other suitable number of days. After the delay period, the subject is once again tested using the same pairs of stimuli used to establish the criterion. In some embodiments, these tests are repeated under circumstances identical, similar, or different to those used in establishing the criterion. Moreover, the subject's performance may be evaluated by establishing a long term success measurement in any suitable manner. In some embodiments, the manner of establishing the long term success measurement may be similar to that described with respect to establishing a criterion, albeit using a generally shorter time period such that the long term success measurement is indicative of the subject's memory, rather than it having relearned the relevant criteria. The subject could then be evaluated by comparing the long term success measurement with the criterion. Statistical or similar analysis may be used to evaluate such results.
In some embodiments, the methods of the present invention may be used to evaluate the efficacy of a potential memory agent and/or a potential training protocol. For example, the methods of the present invention may be used to identify a reduction in the amount of training required to reach a chosen performance level. In such embodiments, a subject may first be evaluated in the absence of a memory agent and subsequently evaluated having been administered a memory agent. Also, a control group of subjects could be tested using certain pairs of stimuli and those results compared to a group of subjects having been administered a given dose of a memory agent. The evaluation of the memory agent and/or training protocol may take a variety of forms. For example, a memory agent may reduce the time and/or number of days of training sessions required to establish or meet a certain criterion, or any other suitable measure of efficacy.
In some embodiments of the present invention, the memory agent is evaluated to see if it alters (e.g., reduces or increases) the number of days or training sessions to reach a criterion of performance. As used herein, the term “criterion” refers to a metric of a desired result and/or a desired level of performance such as, for example, a percentage of correct responses (e.g., correctly paired stimuli) from a test subject or group of test subjects. The criterion may be changed or chosen as desired by the administrator of the protocol, tests, tasks, and/or other embodiments of the present invention. The complexity of the training administered in these embodiments may be manipulated by, for example, changing the criterion, the numbers and/or types stimuli to be paired, the number of days of training, the quantity of training, and/or the type of training. In further embodiments, the training, with or without the memory agent, is sufficient to produce a CREB-dependent change in long term memory, for example a CREB-dependent enhancement of long term memory.
In some embodiments, the methods of the present invention may be used to evaluate the efficacy of a memory impairer. Such embodiments may involve steps similar to or identical to the methods for evaluating a memory agent. However, in such an embodiment, the evaluated indicia would be different. For example, the memory impairer may increase the time required to establish a certain criterion, increase the number of training sessions required to reach a criterion, decrease the ratio of the long term success measurement to the criterion, or any may be evaluated in any other suitable manner.
Any suitable memory agent or memory impairer may be provided by any suitable method, via any suitable delivery system and using any suitable delivery schedule and dose. In addition, any suitable training protocol may be used. Exemplary training protocols, memory agents and memory impairers are specifically discussed in the following sections.
The present invention also includes systems, including systems for use along with the methods disclosed herein. For example, in some embodiments, the system may include all elements necessary to perform an embodiment of the methods of the present invention. Systems of the present invention may be useful for selecting a memory agent as a drug candidate, assessing the effectiveness of a memory agent, assessing the effectiveness of a training protocol, testing a memory agent as a long term memory enhancer, testing a memory agent as a impairer of specific or general memories, and/or testing a memory agent along with a training protocol as a long term memory enhancer. Such systems may involve use of sets of stimuli as described above, including paired stimuli, and testing schedules and protocols as described above.

Training Protocols

As described herein, assessment using the methods and systems of the present invention can comprise two parts: (1) a specific training protocol and (2) administration of a memory agent. In some embodiments, only the training protocol is used. In other embodiments, both training and administration of a memory agent occur. This combination can augment training protocols by reducing the number of days of training sessions required to yield a performance gain relative to that obtained with training alone or by requiring shorter or no rest intervals between training sessions to yield a performance gain. This combination can also augment training protocols by reducing the duration and/or number of training sessions required for the induction in a specific neuronal circuit(s) of a pattern of neuronal activity or by reducing the duration and/or number of training sessions or underlying pattern of neuronal activity required to induce cyclic AMP response element binding protein (CREB)-dependent long-term structural/function (i.e., long-lasting) change among synaptic connections of the neuronal circuit. In this manner, one can improve the efficiency of existing training protocols, thereby yielding significant functional and economic benefits.
Training protocols (e.g., massed training, spaced training) are employed in learning a new language or in learning to play a new musical instrument. Administration of a memory agent in conjunction with training reduces the time and/or number of training sessions required to yield a gain in performance. As a result, less practice (training sessions) is required to learn the new language or to learn to play the new musical instrument.
Training protocols are employed for repeated stimulation of neuronal activity or a pattern of neuronal activity underlying (a) specific neuronal circuit(s) in individuals. Administration of a memory agent in conjunction with training reduces the time and/or number of training sessions and/or underlying pattern of neuronal activity required to achieve a given result, such as induction of CREB-dependent long-term structure/function (i.e., long-lasting) change among synaptic connections of the neuronal circuit.
These methods can enhance specific aspects of memory in an animal (particularly a human or other mammal or vertebrate) in need thereof by (a) administering to the animal a memory agent, for example one that enhances CREB pathway function; and (b) training the animal under conditions sufficient to produce an improvement in performance of a memory task of interest by the animal. In some embodiments, the memory agent is a CREB pathway-enhancing drug.
The described methods can be used to treat a memory deficit associated with a central nervous system (CNS) disorder or condition in an animal in need of said treatment by (a) administering to the animal a memory agent, for example one that enhances CREB pathway function; and (b) training the animal under conditions sufficient to produce an improvement in performance of a particular memory task by the animal. CNS disorders and conditions include age-associated memory impairment, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease (chorea), other senile dementia), psychiatric diseases (e.g., depression, schizophrenia, autism, attention deficit disorder), trauma dependent loss of function (e.g., cerebrovascular diseases (e.g., stroke, ischemia), brain tumor, head or brain injury), genetic defects (e.g., Rubinstein-Taybi syndrome, down syndrome), learning disabilities, and/or combinations thereof.
The described methods can also be used for repeated stimulation of neuronal activity or a pattern of neuronal activity, such as that underlying a specific neuronal circuit(s), in an animal comprising (a) administering to the animal a memory agent, for example one that enhances CREB pathway function; and (b) training the animal under conditions sufficient to stimulate or induce neuronal activity or a pattern of neuronal activity in the animal.
For many tasks in many species, including human, spaced training protocols (multiple training sessions with a rest interval between each) produce stronger, longer-lasting memory than massed training protocols (multiple training sessions with no rest interval in between). Behavior-genetic studies of Pavlovian olfactory learning in Drosophila have established that massed training produces a long-lasting memory that nevertheless decays away in at least four days, is not protein synthesis-dependent, is not disrupted by over-expression of a CREB-repressor transgene, and is disrupted in radish mutants (Tully, T. et al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)). In contrast, spaced training produces a long-lasting memory that persists for at least seven days, is protein synthesis-dependent, is disrupted by over-expression of a CREB-repressor transgene and is normal in radish mutants (Tully, T. et al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)). One day after spaced training, memory retention is composed of both the protein synthesis- and CREB-independent early memory (ARM) and the protein synthesis- and CREB-dependent long-term memory (LTM). Additional massed training is insufficient to induce LTM (Tully, T. et al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).
A growing body of evidence extends these results from invertebrates to mammals. For example, in Aplysia, molecular manipulations of CREB expression, similar to those in flies, suppress or enhance (i) LTM of a facilitatory electrophysiological response at a sensorimotor monosynapse in cell culture and (ii) the synaptic connections between sensory and motor neurons that are normally produced after spaced applications of the facilitatory stimulus (Bartsch, D. et al, Cell, 83(6):979-992 (1995)). In rats, injections of antisense RNA oligonucleotides into hippocampus or amygdala block LTM formation of two different tasks that are dependent on activity in these anatomical regions, respectively (Guzowski, J. F. et al., Proc. Natl. Acad. Sci. USA, 94(6):2693-2698 (1997); and Lamprecht, R. et al., J. Neurosci., 17(21):8443-8450 (1997)). In mice, LTM formation for both implicit and explicit tasks is defective in CREB mutant mice (Bourtchuladze, R. et al., Cell, 79(1):59-68 (1994)).
Training of transgenic mice, carrying a CRE-dependent reporter gene (betagalactosidase), in hippocampal-dependent contextual fear conditioning or passive avoidance tasks induces CRE-dependent reporter gene expression in areas CA1 and CA3 of the hippocampus. Training of these mice in an amygdala-dependent fear conditioning task induces CRE-dependent reporter gene expression in the amygdala, but not the hippocampus. Thus, training protocols that induce LTM formation also induce CRE-dependent gene transcription in specific anatomical areas of the mammalian brain (Impey, S. et al., Nat. Neurosci., 1(7):595-601 (1998)).
With these animal models, three salient cases of LTM enhancement have been demonstrated. First, over-expression of a CREB-activator transgene abrogates the requirements for multiple, spaced training sessions and, instead, induces LTM formation after only one training session (which normally produces little or no memory retention 24 hours later (Yin, J. C. et al, Cell, 81(1):107-115 (1995)). Second, injection of a virally expressed CREB-activator transgene into rat amygdala also is sufficient to enhance memory after massed training for the fear-potentiated startle response, which abrogates the requirement for a rest interval in spaced training (Josselyn, S. A. et al., Society for Neuroscience, Vol. 24, Abstract 365.10 (1998)). Third, LTM formation in CREB-deficient mice (Bourtchuladze, R. et al., Cell, 79(1):59-68 (1994)) can form normally, if mutant mice are subjected to a different, spaced training protocol (Kogan, J. H. et al., Curr. Biol., 7(1):1-11 (1997)).
CREB also appears involved in various forms of developmental and cellular plasticity in the vertebrate brain. For example, neuronal activity increases CREB activity in the cortex (Moore, A. N. et al., J. Biol. Chem., 271(24):14214-14220 (1996)). CREB also mediates developmental plasticity in the hippocampus (Murphy, D. D. et al., Proc. Natl. Acad. Sci. USA, 94(4):1482-1487 (1997)), in the somatosensory cortex (Glazewski, S. et al., Cereb. Cortex, 9(3):249-256 (1999)), in the striatum (Liu, F. C. et al., Neuron, 17(6):1133-1144 (1996)), and in the visual cortex (Pham, T. A. et al., Neuron, 22(1):63-72 (1999)).
CREB appears to be affected in human neurodegenerative disease and brain injury. For example, CREB activation and/or expression is disrupted in Alzheimer's disease (Ikezu, T. et al., EMBO J., 15(10):2468-2475 (1996); Sato, N. et al., Biochem. Biophys. Res. Commun., 232(3):637-642 (1997); and Yamamoto-Sasaki, M. et al., Brain. Res., 824(2):300-303 (1999). CREB activation and/or expression is also elevated after seizures or ischemia (Blendy, J. A. et al., Brain Res., 681(1-2):8-14 (1995); and Tanaka, K. et al., Neuroreport, 10(11):2245-2250 (1999)). “Environmental enrichment” is neuroprotective, preventing cell death by acting through CREB (Young, D. et al., Nat. Med., 5(4):448-453 (1999)).
CREB functions during drug sensitivity and withdrawal. For example, CREB is affected by ethanol (Pandey, S. C. et al., Alcohol Clin. Exp. Res., 23(9):1425-1434 (1999); Constantinescu, A. et al., J. Biol. Chem., 274(38):26985-26991 (1999); Yang, X. et al., Alcohol Clin. Exp. Res., 22(2):382-390 (1998); Yang, X. et al., J. Neurochem., 70(1):224-232 (1998); and Moore, M. S. et al., Cell, 93(6):997-1007 (1998)), by cocaine (Carlezon, W. A., Jr. et al., Science, 282(5397):2272-2275 (1998)), by morphine (Widnell, K. L. et al., J. Pharmacol. Exp. Ther., 276(1):306-315 (1996)), by methamphetamine (Muratake, T. et al., Ann N.Y. Acad. Sci., 844:21-26 (1998)) and by cannabinoid (Calandra, B. et al., Eur. J. Pharmacol., 374(3):445-455 (1999); and Herring, A. C. et al., Biochem. Pharmacol., 55(7):1013-1023 (1998)).
A signal transduction pathway that can stimulate the CREB/CRE transcriptional pathway is the cAMP regulatory system. Consistent with this, mice lacking both adenylate cyclase 1 (AC1) and AC8 enzymes fail to learn (Wong S. T. et al., Neuron, 23(4):787-798 (1999)). In these mice, administration of forskolin to area CA1 of the hippocampus restores learning and memory of hippocampal-dependent tasks. Furthermore, treatment of aged rats with drugs that elevate cAMP levels (such as rolipram and D1 receptor agonists) ameliorates an age-dependent loss of hippocampal-dependent memory and cellular long-term potentiation (Barad, M. et al., Proc. Natl. Acad. Sci. USA, 95(25):15020-15025 (1998)). These latter data suggest that a cAMP signaling is defective in learning-impaired aged rats (Bach, M. E. et al., Proc. Natl. Acad. Sci. USA, 96(9):5280-5285 (1999)).
A growing body of evidence suggests that neurons continue to proliferate in the adult brain (Arsenijevic, Y. et al., Exp. Neurol., 170: 48-62 (2001); Vescovi, A. L. et al., Biomed. Pharmacother., 55:201-205 (2001); Cameron, H. A. and McKay, R. D., J. Comp. Neurol., 435:406-417 (2001); and Geuna, S. et al., Anat. Rec., 265:132-141 (2001)) and that such proliferation is in response to various experiences (Nilsson, M. et al., J. Neurobiol., 39:569-578 (1999); Gould, E. et al., Trends Cogn. Sci., 3:186-192 (1999); Fuchs, E. and Gould, E., Eur. J. Neurosci., 12: 2211-2214 (2000); Gould, E. et al., Biol. Psychiatry, 48:715-720 (2000); and Gould, E. et al., Nat. Neurosci., 2:260-265 (1999)). Experimental strategies now are underway to transplant neuronal stem into adult brain for various therapeutic indications (Kurimoto, Y. et al., Neurosci. Lett., 306:57-60 (2001); Singh, G., Neuropathology, 21:110-114 (2001); and Cameron, H. A. and McKay, R. D., Nat. Neurosci., 2:894-897 (1999)). Much already is known about neurogenesis in embryonic stages of development (Saitoe, M. and Tully, T., “Making connections between synaptic and behavioral plasticity in Drosophila”, In Toward a Theory of Neuroplasticity, J. McEachern and C. Shaw, Eds. (New York: Psychology Press.), pp. 193-220 (2000)). Neuronal differentiation, neurite extension and initial synaptic target recognition all appear to occur in an activity-independent fashion. Subsequent synaptogenesis and synaptic growth, however, then requires ongoing neuronal activity to fine-tune synaptic connections in a functionally relevant manner. These findings suggest that functional (final) integration of transplanted neural stem cells require neuronal activity. Thus, ACT can be used to exercise appropriate neuronal circuits to fine-tune the synaptic connections of newly acquired, transplanted stem cells that differentiate into neurons. By “exercise appropriate neuronal circuit(s)” is meant the induction in the appropriate neuronal circuit(s) of a pattern of neuronal activity, which corresponds to that produced by a particular cognitive training protocol. The cognitive training protocol can be used to induce such neuronal activity. Alternatively, neuronal activity can be induced by direct electrical stimulation of the neuronal circuitry. “Neuronal activity” and “neural activity” are used interchangeably herein.
Memory agents, for example CREB pathway-enhancing drugs, may enhance CREB pathway function, which is required to consolidate newly acquired information into LTM. By “enhance CREB pathway function” is meant the ability to enhance or improve CREB-dependent gene expression. CREB-dependent gene expression can be enhanced or improved by increasing endogenous CREB production, for example by directly or indirectly stimulating the endogenous gene to produce increased amounts of CREB, or by increasing functional (biologically active) CREB. See, e.g., U.S. Pat. No. 5,929,223; U.S. Pat. No. 6,051,559; and International Publication No. WO9611270 (published Apr. 18, 1996), which references are incorporated herein in their entirety by reference. Administration of a memory agent may decrease the training needed to yield a performance gain relative to that yielded with training alone. By “performance gain” is meant an improvement in an aspect of memory performance.
The invention provides methods for evaluating, testing and/or identifying the enhancement of a specific aspect of memory in an animal (particularly in a human or other mammal or vertebrate) (in some embodiments, in need thereof) wherein the enhancement may be due to the (a) administering to the animal a memory agent, for example one that enhances CREB pathway function; and (b) training the animal under conditions sufficient to produce an improvement in performance of a particular memory task by the animal.
Training can comprise one or multiple training sessions and is training appropriate to produce an improvement in performance of the memory task of interest. For example, if an improvement in language acquisition is desired, training would focus on language acquisition. If an improvement in ability to learn to play a musical instrument is desired, training would focus on learning to play the musical instrument. If an improvement in a particular motor skill is desired, training would focus on acquisition of the particular motor skill. The specific memory task of interest is matched with appropriate training.
The invention also provides methods for evaluating, testing and/or identifying the enhancement of memory performance in an animal caused by repeated stimulation of neuronal activity or a pattern of neuronal activity, such as that underlying a specific neuronal circuit(s), in an animal comprising (a) administering to the animal a memory agent which enhances CREB pathway function; and (b) training the animal under conditions sufficient to stimulate or induce neuronal activity or a pattern of neuronal activity in the animal. In this case, training refers to training appropriate to stimulate or induce neuronal activity or a pattern of neuronal activity in the animal.
By “multiple training sessions” is meant two or more training sessions. The memory agent can be administered before, during or after one or more of the training sessions. In a particular embodiment, the memory agent is administered before and during each training session.
Certain training protocols that can be evaluated in accordance with the present invention are known and readily available in the art. See for example, Karni, A. and Sagi, D., “Where practice makes perfect in text discrimination: evidence for primary visual cortex plasticity”, Proc. Natl. Acad. Sci. USA, 88:4966-4970 (1991); Karni, A. and Sagi, D., “The time course of learning a visual skill,” Nature, 365:250-252 (1993); Kramer, A. F. et al., “Task coordination and aging: explorations of executive control processes in the task switching paradigm,” Acta Psychol. (Amst.), 101:339-378 (1999); Kramer, A. F. et al., “Training for executive control: Task coordination strategies and aging,” In Aging and Skilled Performance: Advances In Theory and Applications, W. Rogers et al., eds. (Hillsdale, N.J.: Erlbaum) (1999); Rider, R. A. and Abdulahad, D. T., “Effects of massed versus distributed practice on gross and fine motor proficiency of educable mentally handicapped adolescents,” Percept. Mot. Skills, 73:219-224 (1991); Willis, S. L. and Schaie, K. W., “Training the elderly on the ability factors of spatial orientation and inductive reasoning,” Psychol. Aging, 1:239-247 (1986); Willis, S. L. and Nesselroade, C. S., “Long-term effects of fluid ability training in old-old age,” Develop. Psychol., 26:905-910 (1990); Wek, S. R. and Husak, W. S., “Distributed and massed practice effects on motor performance and learning of autistic children,” Percept. Mot. Skills, 68:107-113 (1989); Verhaehen, P. et al., “Improving memory performance in the aged through mnemonic training: a meta-analytic study,” Psychol. Aging, 7:242-251 (1992); Verhaeghen, P. and Salthouse, T. A., “Meta-analyses of age-cognition relations in adulthood: estimates of linear and nonlinear age effects and structural models,” Psychol. Bull., 122:231-249 (1997); Dean, C. M. et al., “Task-related circuit training improves performance of locomotor tasks in chronic stroke: a randomized, controlled pilot trial,” Arch. Phys. Med. Rehabil., 81:409-417 (2000); Greener, J. et al., “Speech and language therapy for aphasia following stroke,” Cochrane Database Syst. Rev., CD000425 (2000); Hummelsheim, H. and Eickhof, C., “Repetitive sensorimotor training for arm and hand in a patient with locked-in syndrome,” Scand. J. Rehabil. Med., 31:250-256 (1999); Johansson, B. B., “Brain plasticity and stroke rehabilitation. The Willis lecture,” Stroke, 31:223-230 (2000); Ko Ko, C., “Effectiveness of rehabilitation for multiple sclerosis,” Clin. Rehabil., 13 (Suppl. 1):33-41 (1999); Lange, G. et al., “Organizational strategy influence on visual memory performance after stroke: cortical/subcortical and left/right hemisphere contrasts,” Arch. Phys. Med. Rehabil., 81:89-94 (2000); Liepert, J. et al., “Treatment-induced cortical reorganization after stroke in humans,” Stroke, 31:1210-1216 (2000); Lotery, A. J. et al., “Correctable visual impairment in stroke rehabilitation patients,” Age Ageing, 29:221-222 (2000); Majid, M. J. et al., “Cognitive rehabilitation for memory deficits following stroke” (Cochrane review), Cochrane Database Syst. Rev., CD002293 (2000); Merzenich, M. et al., “Cortical plasticity underlying perceptual, motor, and cognitive skill development: implications for neurorehabilitation,” Cold Spring Harb. Symp. Quant. Biol., 61:1-8 (1996); Merzenich, M. M. et al., “Temporal processing deficits of language-learning impaired children ameliorated by training,” Science, 271:77-81 (1996); Murphy, E., “Stroke rehabilitation,” J. R. Coll. Physicians Lond., 33:466-468 (1999); Nagarajan, S. S. et al., “Speech modifications algorithms used for training language learning-impaired children,” IEEE Trans. Rehabil. Eng., 6:257-268. (1998); Oddone, E. et al., “Quality Enhancement Research Initiative in stroke: prevention, treatment, and rehabilitation,” Med. Care 38:192-1104 (2000); Rice-Oxley, M. and Turner-Stokes, L., “Effectiveness of brain injury rehabilitation,” Clin. Rehabil., 13(Suppl 1):7-24 (1999); Tallal, P. et al., “Language learning impairments: integrating basic science, technology, and remediation,” Exp. Brain Res., 123:210-219 (1998); Tallal, P. et al., “Language comprehension in language-learning impaired children improved with acoustically modified speech,” Science, 271:81-84 (1996), which references are incorporated herein in their entirety by reference.
Accordingly, the invention also relates to methods of enhancing long term memory in an animal (particularly in a human or other mammal or vertebrate) in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer, and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
In one embodiment, the invention also relates to a method of treating an age-associated memory impairment in an animal in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
In another embodiment, the invention relates to a method of treating a memory deficit associated with a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, other senile dementia) in an animal in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
In another embodiment, the invention relates to a method of treating a memory deficit associated with a psychiatric disease (e.g., depression, schizophrenia, autism, attention deficit disorder) in an animal in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
In another embodiment, the invention relates to a method of treating a memory deficit associated with trauma dependent loss of memory function (e.g., cerebrovascular diseases (e.g., stroke, ischemia), brain tumor, head or brain injury) in an animal in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
In another embodiment, the invention relates to a method of treating a memory deficit associated with a genetic defect (e.g., Rubinstein-Taybi syndrome, down syndrome) in an animal in need of said treatment comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.
The invention also relates to methods of therapy of a memory deficit associated with a CNS disorder or condition in an animal having undergone neuronal stem cell manipulation (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal. By “neuronal stem cell manipulation” is meant that (1) exogenous neuronal stem cells are transplanted into the brain or spinal chord of an animal or (2) endogenous neuronal stem cells are stimulated or induced to proliferate in the animal. Methods of transplanting neuronal stem cells into the brain or spinal chord of an animal are known and readily available in the art (see, e.g., Cameron, H. A. and McKay, R. D., Nat. Neurosci., 2:894-897 (1999); Kurimoto, Y. et al., Neurosci. Lett., 306:57-60 (2001); and Singh, G., Neuropathology, 21:110-114 (2001)). Methods of stimulating or inducing proliferation of endogenous neuronal stem cells in an animal are known and readily available in the art (see, e.g., Gould, E. et al., Trends Cogn. Sci, 3:186-192 (1999); Gould, E. et al., Biol. Psychiatry, 48:715-20 (2000); Nilsson, M. et al, J. Neurobiol., 39:569-578 (1999); Fuchs, E. and Gould, E., Eur. J. Neurosci., 12:2211-2214 (2000); and Gould, E. et al., Nat. Neurosci., 2:260-265 (1999)). The particular methods of transplanting neuronal stem cells into the brain or spinal chord of an animal and the particular methods of stimulating or inducing proliferation of endogenous neuronal stem cells in an animal are not critical to the practice of the invention.
The invention further relates to methods of improving or enhancing memory and/or performance in an animal with a learning, language or reading disability, or combinations of any of the foregoing, comprising (a) administering to the animal a memory agent identified, tested or evaluated by the methods of assessing, evaluating and/or testing the effectiveness of a memory agent, selecting a memory agent as a drug compound, or testing a memory agent as a long term memory enhancer; and (b) training the animal under conditions sufficient to enhance long term memory in the animal.

Memory Agents

Memory agents, as used herein, are compounds with pharmacological activity relative to memory disorders and/or memory dysfunctions, and include drugs, chemical compounds, ionic compounds, organic compounds, organic ligands, including cofactors, saccharides, recombinant and synthetic peptides, proteins, peptoids, nucleic acid sequences, including genes, nucleic acid products, and other molecules and compositions. Memory agents may, for example, enhance or impair short or long term memory. For example, memory agents may be any of the compounds set forth in U.S. patent application Ser. No. 11/679,782 entitled “Therapeutic piperazones” and filed on Feb. 27, 2007; U.S. patent application Ser. No. 11/608,746 entitled “Indolone Compounds Useful To Treat Cognitive Impairment” and filed on Dec. 8, 2006; and U.S. patent application Ser. No. 11/679,775 entitled “Therapeutic Compounds” and filed on Feb. 27, 2006, U.S. Provisional Patent Application Ser. No. 60/942,992, entitled “Therapeutic Pyrazoloquinoline Urea Derivatives” filed on Jun. 8, 2007, each of the above patent applications is hereby expressly incorporated by reference herein in its entirety.
A memory impairer, as used herein, are compounds with pharmacological activity relating to retained memories (for example, post-traumatic stress disorder), and include drugs, chemical compounds, ionic compounds, organic compounds, organic ligands, including cofactors, saccharides, recombinant and synthetic peptides, proteins, peptoids, nucleic acid sequences, including genes, nucleic acid products, and other molecules and compositions. The memory impairers and memory enhancers of the present invention can be used with any of the present methods and systems.
For example, memory agents can be cell permeant cAMP analogs (e.g., 8-bromo cAMP); activators of adenylate cyclase 1 (AC1) (e.g., forskolin); agents affecting G-protein linked receptor, such as, but not limited to adrenergic receptors and opioid receptors and their ligands (e.g., phenethylamines); modulators of intracellular calcium concentration (e.g., thapsigargin, N-methyl-D-aspartate (NMDA) receptor agonists); inhibitors of the phosphodiesterases responsible for cAMP breakdown (e.g., rolipram (which inhibits phosphodiesterase 4), iso-buto-metho-xanthine (IBMX) (which inhibits phosphodiesterases 1 and 2)); modulators of protein kinases and/or protein phosphatases, which mediate CREB protein activation and CREB-dependent gene expression. Memory agents can also be exogenous CREB, CREB analogs, CREB-like molecules, biologically active CREB fragments, CREB fusion proteins, and/or nucleic acid sequences encoding exogenous CREB, CREB analogs, CREB-like molecules, biologically active CREB fragments and/or CREB fusion proteins.
Memory agents can also be CREB function modulators, and/or nucleic acid sequences encoding CREB function modulators. CREB function modulators, as used herein, have the ability to modulate CREB pathway function. By “modulate” is meant the ability to change (increase or decrease) or alter CREB pathway function.
Memory agents can be compounds which are capable of enhancing CREB function in the CNS. Such compounds include, but are not limited to, compounds which affect membrane stability and fluidity and specific immunostimulation. In a particular embodiment, the memory agent is capable of transiently enhancing CREB pathway function in the CNS.
CREB analogs, or derivatives, are defined herein as proteins having amino acid sequences analogous to endogenous CREB. Analogous amino acid sequences are defined herein to mean amino acid sequences with sufficient identity of amino acid sequence of endogenous CREB to possess the biological activity of endogenous CREB, but with one or more “silent” changes in the amino acid sequence. CREB analogs include mammalian CREM, mammalian ATF-1 and other CREB/CREM/ATF-1 subfamily members.
CREB-like molecule, as the term is used herein, refers to a protein which functionally resembles (mimics) CREB. CREB-like molecules need not have amino acid sequences analogous to endogenous CREB.
Examples of memory agents can include:
Examples of memory agents can also include:
wherein “Me” means methyl and “cPent” means “cyclopentyl.” The above formula embraces both enantiomers and mixtures thereof. In a particular embodiment, (HT-0712), the 3 and 5 carbons of the above formula are in the S configuration.
Examples may also include a compound having the structure:
wherein each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl, or C₅-C₇cycloalkenyl; —F, —Cl, —Br, or —I; —NO₂; —N₃; —CN; —OR₄, —OCOR₄, —COR₄, —NCOR₄, —N(R₄)₂, —CON(R₄)₂, or —COOR₄; aryl or heteroaryl; or any two of Y₁, Y₂, Y₃and Y₄present on adjacent carbon atoms can constitute a methylenedioxy group; wherein each R₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl, C₅-C₇cycloalkenyl, aryl or aryl(C₁-C₆)alkyl; wherein A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; wherein A′ is
wherein R₁and R₂are each independently H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, or —CN; wherein R₃is H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, —CN, —OR₆, aryl or heteroaryl; wherein R₅is straight chained or branched C₁-C₇alkyl, —N(R₄)₂, —OR₄or aryl; wherein R₆is straight chained or branched C₁-C₇alkyl or aryl; wherein B is C₃-C₇cycloalkyl, C₅-C₇cycloalkenyl, adamantyl, aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-naphthridinyl, pteridinyl, or phthalimidyl; provided however, if B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo [b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-napthyridinyl, pteridinyl, or phthalimidyl the carbon atom or carbon atoms ortho to the nitrogen atom of the imine bond may only be substituted with one or more of the following —F, —Cl, —Br, —I, —CN, methyl, ethyl or methoxy; wherein n is an integer from 1 to 4 inclusive, and wherein aryl is phenyl or napthyl, including phenyl and napthyl substituted with one or more of the following: —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkenyl, C₃C₇cycloalkyl, C₃-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —OR₄, SR₄, —OCOR₄, —COR₄, —NCOR₄, —CO₂R₄, —CON(R₄)₂or (CH₂)_n—O—(CH₂)_m.—CH₃.
In some embodiments A is aryl, heteroaryl, or heteroaryl (C₁-C₆)alkyl; and wherein aryl is substituted with —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkynyl, C₃-C₇cycloalkyl, C₃-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —N(R₄)₂, —OR₄, —SR₄, —OCOR₄, —COR₄, —NCOR₄, —CO₂R₄, —CON(R₄)₂or —(CH₂)_n—O—(CH₂)_mCH₃. A may also be aryl, heteroaryl, heteroaryl(C₁-C₆)alkyl or —(CH₂)_n—CC—R₄; wherein the aryl is substituted with —OH. In other embodiments, wherein A is A′ and A′ is
In other embodiments each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, —CF₃, —F, —C₁, —Br, —I, —OR₄, —N(R₄)₂, or —CON(R₄)₂; wherein each R₄is independently —H; straight chained or branched C₁-C₇alkyl, —CF₃, or phenyl; wherein A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; and wherein A′ is
In certain embodiments, B is C₃-C₇cycloalkyl or adamantyl. In other embodiments, B is pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-napthyridinyl, pteridinyl, or phthalimidyl. In other embodiments B is aryl or is phenyl and the phenyl is optionally substituted with one or more of the following: —F, —Cl, —Br, —CF₃, straight chained or branched C₁-C₇alkyl, —OR₄, —COR₄, —NCOR₄, —CO₂R₄, or —CON(R₄)₂.
Further examples include compounds have the following structures:
Further examples include a compound having the structure:
wherein each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl or C₅-C₇cycloalkenyl; —F, —Cl, —Br, or —I; —NO₂; —N₃; —CN; —OR₄, —SR₄, —OCOR₄, —COR₄, —NCOR₄, —N(R₄)₂, —CON(R₄)₂, or —COOR₄; aryl or heteroaryl; or any two of Y₁, Y₂, Y₃and Y₄present on adjacent carbon atoms can constitute a methylenedioxy group; wherein each R₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl, C₅-C₇cycloalkenyl, aryl or aryl(C₁-C₆)alkyl; wherein A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; wherein A′ is
wherein R₁and R₂are each independently H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, or —CN; wherein R₃is H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, —CN, —OR₆, aryl or heteroaryl; wherein R₅is straight chained or branched C₁-C₇alkyl, —N(R₄)₂, —OR₄or aryl; wherein R₆is straight chained or branched C₁-C₇alkyl or aryl; wherein B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-napthyridinyl, pteridinyl, or phthalimidyl; provided however, that the carbon atom or carbon atoms ortho to the nitrogen atom of the imine bond may only be substituted with one or more of the following —F, —Cl, —Br, —I, —CN, methyl, ethyl or methoxy; wherein n is an integer from 1 to 4 inclusive; wherein aryl is phenyl or napthyl, including phenyl and napthyl substituted with one or more of the following: —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkynyl, C₃-C₇cycloalkyl, C₁-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —OR₄, SR₄, —OCOR₄, —COR₄, —NCOR₄, —CO₂R₄, —CON(R₄)₂or (CH₂)_n—O—(CH₂)_m—CH₃; or a pharmaceutically acceptable salt thereof. In some embodiments, each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, —CF₃, —F, —Cl, —Br, —I, —OR₄, —N(R₄)₂, or —CON(R₄)₂.
In some embodiments, A is aryl, heteroaryl, heteroaryl(C₁-C₆)alkyl or —(CH₂)_n—CC—R₄; wherein the aryl is substituted with —OH. In other embodiments, A is aryl, heteroaryl, or heteroaryl(C₁-C₆)alkyl; and wherein aryl is substituted with —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkynyl, C₃-C₇cycloalkyl, C₃-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —N(R₄)₂, —OR₄, —SR₄, —OCOR₄, —COR₄, —CO₂R₄, —CON(R₄)₂or —(CH₂)_n—O—(CH₂)_mCH₃. A may also be aryl or aryl(C₁-C₆)alkyl. In other embodiments, A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; and A′ is
The compounds described herein may be enantiomerically and/or diastereomerically pure. The may also be a pure Z imine isomer or a pure Z alkene isome, or a pure E imine isomer or a pure E alkene isomer.
Additional examples include:
Additional examples may include a compound of the following formula:
or a pharmaceutically acceptable salt thereof wherein:
R₁, R₂, R_d, and R₄are each independently selected from the group consisting of hydrogen, hydroxy, halo, cyano, —CONR_aR_b, —NR_aR_b, hydroxy(C₁-C₆)alkyl, aryl, heteroaryl, heterocycle, amino(C₁-C₆)alkyl, (C₁-C₆)alkyl optionally substituted with up to 5 fluoro, and (C₁-C₆)alkoxy optionally substituted with up to 5 fluoro;
each R_aand R_bare independently hydrogen, (C₁-C₆)alkyl, aryl, (C₁-C₆)alkylOC(O)—, or arylOC(O)—, or R_aand R_bare taken together with the nitrogen to which they are attached to form a heterocycle group optionally substituted with one or more R_d; wherein the heterocycle group optionally include one or more groups selected from 0 (oxygen), S(O)_z, and NR_c; each z is an integer selected from 0, 1, and 2;
each R_cis independently hydrogen, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —C(O)O(C₁-C₆)alkyl, —C(O)Oaryl, (C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)alkylO(CH₂)_m, hydroxy(C₁-C₆)alkyl, aryl, heteroaryl, Heterocycle, arylO(C₁-C₆)alkyl, —C(O)NR_g(C₁-C₆)alkyl, —C(O)NR_garyl, —S(O),(C₁-C₆)alkyl, —S(O), aryl, —C(O)(C₁-C₆)alkyl, arylC(O)—, (C₁-C₆)alkyl optionally substituted with up to 5 fluoro, or (C₁-C₆)alkoxy optionally substituted with up to 5 fluoro;
each m is an integer selected from 2, 3, 4, 5, and 6;
each R_dis independently selected from the group consisting of hydrogen, halo, oxo, hydroxy, —C(O)NR_aR_b, —NR_aR_b, hydroxy(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, (C₁-C₆)alkyl optionally substituted with up to 5 fluoro, and (C₁-C₆)alkoxy optionally substituted with up to 5 fluoro; R_cand R_rare each independently selected from the group consisting of hydrogen, (C₁-C₆)alkyl, aryl, —S(O),(C₁-C₆)alkyl, —S(O),aryl, —CONRg(C₁-C₆alkyl), (C₁-C₆)alkylC(O)—, arylC(O)—, (C₁-C₆)alkylOC(O)—, and arylOC(O)—;
R_gis hydrogen or (C₁-C₆)alkyl;
R₅and R₆are each independently selected from the group consisting of hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, and aryl, or R₅and R₆are taken together with the nitrogen to which they are attached to form a heterocycle group optionally substituted with one or more R_d; wherein the heterocycle group optionally include one or more groups selected from 0 (oxygen), S(O)_z, and NR_c;
R₇is selected from the group consisting of hydrogen, hydroxy, halo, hydroxy(C₁-C₆)alkyl, (C₁-C₆)alkyl optionally substituted with up to 5 fluoro, and (C₁-C₆)alkoxy optionally substituted with up to 5 fluoro;
Ar is aryl, or heteroaryl, each optionally substituted with one or more R₈; and
each R₈is independently hydrogen, halo, CF₃, CF₂H, hydroxy, cyano, nitro,(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, (C₁-C₆)alkoxy, —NR_aR_b, aryl, heteroaryl or heterocycle.
Other examples include the compound of following formula:
and pharmaceutically acceptable salts thereof.
R₅and R₆may, for example, together with the nitrogen to which they are attached, form a piperidinyl, pyrrolidinyl, morpholinyl, or thiomorpholinyl ring in the compound of the above formula.
Yet further examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof, wherein: X is N(R_c), O (oxygen), C(R_d)₂, or S(O)_z; z is an integer selected from 0, 1, and 2; each R_dis independently selected from the group consisting of hydrogen, halo, oxo, hydroxy, —C(O)NR_aR_b, —NR₃R_b, hydroxy(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, (C₁-C₆)alkyl optionally substituted with up to 5 fluoro, and (C₁-C₆)alkoxy optionally substituted with up to 5 fluoro; and n is an integer selected from 0, 1, and 2; with the proviso that when n=0 then X is C(R_d)₂.
Other examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof.
Other examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof, wherein n is 0, 1, or 2.
In some embodiments, R₂is Methyl, fluoro, or OMe. In some embodiments, R₃is Methyl, fluoro, or OMe. In some embodiments, R₂and R₃are fluoro. In another embodiment, R₂and R₃are Methyl.
Other examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof.
Other examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof, wherein each Y is independently N or C(R₈). In some embodiments, R₅and R₆, together with the nitrogen to which they are attached, form a piperidinyl, pyrrolidinyl, morpholinyl, or thiomorpholinyl ring, each optionally substituted with one or more R_d.
Further examples include a compound of the following formula:
and pharmaceutically acceptable salts thereof.
In some embodiments n can be 0, 1 or 2. In some embodiments, R₂may be Methyl, fluoro or OMe. In some embodiments, R₃can be Methyl, fluoro or OMe. In some embodiments, R₂and R₃can be fluoro. In another embodiment, R₂and R₃can be Methyl.
Additional examples include a compounds of the formulas:
and pharmaceutically acceptable salts thereof.
Examples also include a compound of the following formula:
wherein: R₁is H, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl((C₁-C₆)alkyl, halo(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, aryl(C₁-C₆)alkanoyl, het, het(C₁-C₆)alkyl, het(C₁-C₆)alkoxy, or het(C₁-C₆)alkanoyl;
n is 1 or 2;
m is 1 or 2;
W is O, S, or two hydrogens;

X is 0 or N—Y—R₄;

Y is a direct bond, —CH₂—, —C(=0)-, —C(═S)—, —O—, —C(=0)O—, —OC(=0)-, —C(=0)NR_a—, —S—, —S(=0)-, —S(=0)₂—, or —S(=0)₂NR_a—;
R₄is H, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, hydroxy, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonyl, carboxy, aryl, aryl(C₁-C₆)alkyl, het, NR_dR_c, —C(=0)NR_dR_c, NR_dR_c(C₁-C₆)alkyl, or het(C₁-C₆)alkyl;
R_ais H, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₆)alkoxy(C₂-C₆)alkyl, or (C₃-C₈)cycloalkyl(C₁-C₆)alkyl;
Z is a phenyl ring substituted with one or more substituents independently selected from (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, (C₃-C₈)cycloalkyloxy, and (C₃-C₈)cycloalkyl(C₁-C₆)alkoxy; or Z is a phenyl ring that is fused to a saturated, partially unsaturated, or aromatic, mono- or bicyclic ring system comprising from about 3 to about 8 atoms selected from carbon, oxygen, and NR_b, wherein the mono- or bicyclic ring system of Z is optionally substituted with one or more R_b, and wherein the phenyl ring that is fused to the mono- or bicyclic ring system is optionally substituted with one or more substitaents independently selected from (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₂-C₆)alkoxy, (C₃-C₈)cycloalkyloxy, and (C₃-C₈)cycloalkyl(C₁-C₆)alkoxy;
R_bis absent, H, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₆)alkoxy(C₂-C₆)alkyl, or (C₃-C₈)cycloalkyl(C₁-C₆)alkyl;
R_c, is (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, aryl(C₁-C₆)alkanoyl, het, het(C₁-C₆)alkyl, het(C₁-C₆)alkoxy, or het(C₁-C₆)alkanoyl; each R_dand R_cis independently H, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₂-C₆)alkenyloxy, (C₂-C₆)alkynyloxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, NR_fR_g, or aryl(C₁-C₆)alkoxy; and
each R_fand R_gis independently H, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, or aryl(C₁-C₆)alkoxy; or R_fand R_gtogether with the nitrogen to which they are attached form a pyrrolidino, piperidino, piperazino, morpholino, or thiomorpholino ring;
wherein any aryl or het of R₁and R₄is optionally substituted with one or more substitutents independently selected from (C₁-C₆)alkyl, phenyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₃-C₈)cycloalkyloxy, (C₃-C₈)cycloalkyl(C₁-C₆)alkoxy, halo(C₂-C₆)alkoxy, cyan, nitro, halo, carboxy or NR_dR_e;
and wherein the ring containing X is optionally substituted on carbon with one or more halo, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy.
Including any pharmaceutically acceptable salt thereof.
Other examples include a compound of the following formula:
wherein: R′ is (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, or aryl, unsubstituted or substituted with one or more R_e; one of R²and R³is absent and the other is hydrogen, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, amino(C₂-C₆)alkyl, or aryl, each unsubstituted or substituted with one or more groups selected from alkyl, halo, haloalkyl or nitro, Het, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, (C₁-C₆) or Het(C₁-C₆)alkyl;
B is aryl, thiophene, heteroaryl, furan or pyrrole;

X is —C(=0), —C(—S), —C(R⁴)₂, —C(OH)—, or —S(O)_z;

each z is independently 0, 1, or 2;

Y is R⁴—N(R⁴)₂, —OR₄, —SR₄or —C(R⁴)₃;

each R⁴is independently selected from the group consisting of hydrogen, (C₁C₆) alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₂-C₆)alkyl, hydroxy(C₂-C₆)alkyl, cyano(C₁-C₆)alkyl, (C₁-C₆)alkthio(Cz-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, aryloxy(C₂-C₆)alkyl, halo(C₂-C₆)alkyl, (C₁-C₆)alkoxycarbonyl(C₁C₆)alkyl, NR_aR_{b, Het, or Het(C} ₁-C₆)alkyl, unsubstituted or substituted with one or more or two R⁴groups are taken together with the atom to which they are attached to form aryl, Het, or a saturated or unsaturated 3-8 membered monocyclic or 8-12 membered bicyclic ring system comprising carbon atoms and optionally comprising one or more additional heteroatoms selected from O, S(O)_z, and NR_c, wherein each ring system is optionally substituted with one or more R_d;
each R_aand R_bis independently hydrogen or (C₁-C₆)alkyl;
each R_c, is independently hydrogen, aryl, S(O)₂, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl, alkoxy(C₁-C₆)alkyl, Het, (C₁-C₆)alkoxycabonyl or (C₁-C₆)alkyl, unsubstituted or substituted with one or more subtituents R_e;
each R_dis independently halo, hydroxy, cyano, nitro, azido, amino, (C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, amido, (C₁-C₆)alkyamido, aryl amido, carboxylic acid, (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, (C₁-C₆)alkanoyloxy, Het, aryl, Het(C₁-C₆)alkyl, or aryl(C₁-C₆)alkyl, (C₁-C₆)alkylaryl, sulfonyl, sulfonamido, urea, carbamate, unsubstituted or substituted with one or more substituents R_e, or two R_dcome together with the atom to which they are attached to form a ketone or spirocyclic carbocyclic or heterocyclic ring, or two R_dcome together with the atoms to which they are attached to form a bicyclic carbocyclic or heterocyclic ring, wherein each spirocyclic or bicyclic ring is unsubstituted or substituted with one or more halo, hydroxy, cyano, nitro, azido, (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, (C₁-C₆)alkanoyloxy, NR_fR_g, R_fR_gNC(=0)-, phenyl, or phenyl(C₁-C₆)alkyl, sulfonyl, sulfonamido, urea, carbamate, wherein R_fand R_gtogether with the nitrogen to which they are attached form a piperidino, pyrrolidino, morpholino, or thiomorpholino ring, unsubstituted or substituted with one or more substituents R_e;
each R_c, is independently selected from halo, hydroxy, cyano, nitro, azido, (C₁-C₆)alkyl, Het, aryl, (C₁-C₆)alkylHet, (C₁-C₆)alkylaryl, (C₁-C₆)alkylHet(C₁-C₆)alkyl, (C₁-C₆)alkylaryl(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, and (C₁-C₆)alkanoyloxy;
R⁵is H, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl, aryl(C₁-C₆)alkyl; and
each R⁶is H, (C₁-C₆)alkyl, amino, amido, keto, or aryl(C₁-C₆)alkyl, with the proviso that when B is thiophene, R¹is trifluoromethyl, R²is methyl, R³and R⁶are absent, R⁵is H, X is C(=0) and Y is N(R⁴)₂, both R⁴are not methyl.
Additional examples include a compound of the following formula:
wherein R¹is (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, or aryl, unsubstituted or substituted with one or more R_c; one of R²and R³is absent and the other is hydrogen, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, amino(C₂-C₆)alkyl, or aryl, each unsubstituted or substituted with one or more groups selected from alkyl, halo, haloalkyl or nitro, Het, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, (C₁-C₆) or Het(C₁-C₆)alkyl;
each of Z¹, Z², and Z³is independently C(R⁶)_p, N(R⁶)_q, O, or S, wherein if Z¹is N(R⁶)_q, O, or S, at least one of Z¹or Z²must be N(R⁶)_q, O, or S;
each p is independently 0, 1, or 2;
each q is independently 0 or 1;

X is —C(=0), —C(═S), —C(R⁴)₂, or —S(O)L;

each z is independently 0, 1, or 2;
Y is R⁴, —N(R⁴)_z, —OR⁴, —SR⁴, or —C(R⁴)₃;
each R⁴is independently selected from the group consisting of hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₂-C₆)alkyl, hydroxy(C₂-C₆)alkyl, cyano(C₁-C₆)alkyl, (C₁-C₆)alkthio(C₂-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, aryloxy(C₂-C₆)alkyl, halo(C₂-C₆)alkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, NR_aR_b, Het, or Het(C₁-C₆)alkyl, wherein each alkyl, aryl, or Het is unsubstituted or substituted with one or more R_d, or two R⁴groups are taken together with the atom to which they are attached to form aryl, Het, or a saturated or unsaturated 3-8 membered monocyclic or 8-12 membered bicyclic ring system comprising carbon atoms and optionally comprising one or more additional heteroatoms selected from O, S(O)_z, and NR_c, wherein each ringsystem is optionally substituted with one or more R_d;
each R_aand R_bis independently hydrogen or (C₁-C₆)alkyl;
each R_cis independently hydrogen, aryl, S(O)₂, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl, alkoxy(C₁-C₆)alkyl, Het, (C₁-C₆)alkoxycabonyl or (C₁-C₆)alkyl, unsubstituted or substituted with one or more substituents R_e;
each R_dis independently halo, hydroxy, cyano, nitro, azido, amino, (C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, amido, (C₁-C₆)alkyamido, aryl amido, carboxylic acid, (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, (C₁-C₆)alkanoyloxy, Het, aryl, Het(C₁-C₆)alkyl, or aryl(C₁-C₆)alkyl, (C₁-C₆)alkylaryl, sulfonyl, sulfonamide, urea, carbamate, unsubstituted or substituted with one or more substituents R_e, or two R_dcome together with the atom to which they are attached to form a ketone or spirocyclic carbocyclic or heterocyclic ring, or two R_dcome together with the atoms to which they are attached to form a bicyclic carbocyclic or heterocyclic ring, wherein each spirocyclic or bicyclic ring is unsubstituted or substituted with one or more halo, hydroxy, cyano, nitro, azido, (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, (C₁-C₆)alkanoyloxy, NR_fR_g, R_fR_gNC(=0)-, phenyl, or phenyl(C₁-C₆)alkyl, sulfonyl, sulfonamido, urea, carbamate, wherein R_fand R_gtogether with the nitrogen to which they are attached form a piperidino, pyrrolidino, morpholino, or thiomorpholino ring, unsubstituted or substituted with one or more substituents R_e;
each R_eis independently selected from halo, hydroxy, cyano, nitro, azido, (C₁-C₆)alkyl, Het, aryl, (C₁-C₆)alkylHet, (C₁-C₆)alkylaryl, (C₁-C₆)alkylHet(C₁-C₆)alkyl, (C₁-C₆)alkylaryl(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, carboxy, and (C₁-C₆)alkanoyloxy;
R⁵is H, (C₁-C₆)alkyl, aryl(C₁-C₆)alkyl; and
each R⁶is H, (C₁-C₆)alkyl, amino, amido, keto, or aryl(C₁-C₆)alkyl; with the proviso that when X is —C(=0), Y is N(R⁴)_z, Z₁is O, Z²is N, and Z₃is CH, both R⁴of Y are not H.
Biologically active polypeptide fragments of CREB can include only a part of the full-length amino acid sequence of CREB, yet possess biological activity. Such fragments can be produced by carboxyl or amino terminal deletions, as well as internal deletions.
Fusion proteins comprise a CREB protein as described herein, referred to as a first moiety, linked to a second moiety not occurring in the CREB protein. The second moiety can be a single amino acid, peptide or polypeptide or other organic moiety, such as, without limitation, a carbohydrate, a lipid or an inorganic molecule.
Nucleic acid sequences are defined herein as heteropolymers of nucleic acid molecules. The nucleic acid molecules can be double stranded or single stranded and can be a deoxyribonucleotide (DNA) molecule, such as cDNA or genomic DNA, or a ribonucleotide (RNA) molecule. As such, the nucleic acid sequence can, for example, include one or more exons, with or without, as appropriate, introns, as well as one or more suitable control sequences. In one example, the nucleic acid molecule contains a single open reading frame which encodes a desired nucleic acid product. The nucleic acid sequence can be operably linked to a suitable promoter.
A nucleic acid sequence encoding a desired CREB protein, CREB analog (including CREM, ATF-1), CREB-like molecule, biologically active CREB fragment, CREB fusion protein or CREB function modulator can be isolated from nature, modified from native sequences or manufactured de novo, as described in, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998); and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York. (1989). Nucleic acids can be isolated and fused together by methods known in the art, such as exploiting and manufacturing compatible cloning or restriction sites.
Typically, the nucleic acid sequence will be a gene which encodes the desired CREB protein, CREB analog, CREB-like molecule, CREB fusion protein or CREB function modulator. Such a gene is typically operably linked to suitable control sequences capable of effecting the expression of the CREB protein or CREB function modulator, preferably in the CNS. The term “operably linked”, as used herein, is defined to mean that the gene (or the nucleic acid sequence) is linked to control sequences in a manner which allows expression of the gene (or the nucleic acid sequence). Generally, but not always, operably linked means contiguous.
Control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable messenger RNA (mRNA) ribosomal binding sites and sequences which control termination of transcription and translation. In a particular embodiment, a recombinant gene (or a nucleic acid sequence) encoding a CREB protein, CREB analog, CREB-like molecule, biologically active CREB fragment, CREB fusion protein or CREB function modulator can be placed under the regulatory control of a promoter which can be induced or repressed, thereby offering a greater degree of control with respect to the level of the product.
As used herein, the term “promoter” refers to a sequence of DNA, usually upstream (5′) of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. Suitable promoters are well known in the art. Exemplary promoters include the SV40 and human elongation factor (EFI). Other suitable promoters are readily available in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); and U.S. Pat. No. 5,681,735).
Memory agents can enhance CREB pathway function by a variety of mechanisms. For example, a memory agent can affect a signal transduction pathway which leads to induction of CREB-dependent gene expression. Induction of CREB-dependent gene expression can be achieved, for example, via up-regulation of positive effectors of CREB function and/or down-regulation of negative effectors of CREB function. Positive effectors of CREB function include adenylate cyclases and CREB activators. Negative effectors of CREB function include cAMP phosphodiesterase (cAMP PDE) and CREB repressors.
A memory agent can enhance CREB pathway function by acting biochemically upstream of or directly acting on an activator or repressor form of a CREB protein and/or on a CREB protein containing transcription complex. For example, CREB pathway function can be affected by increasing CREB protein levels transcriptionally, post-transcriptionally, or both transcriptionally and post-transcriptionally; by altering the affinity of CREB protein to other necessary components of the of the transcription complex, such as, for example, to CREB-binding protein (CBP protein); by altering the affinity of a CREB protein containing transcription complex for DNA CREB responsive elements in the promoter region; or by inducing either passive or active immunity to CREB protein isoforms. The particular mechanism by which a memory agent enhances CREB pathway function is not critical to the practice of the invention.
Memory agents can be administered directly to an animal in a variety of ways. In a specific embodiment, memory agents are administered systemically. Other routes of administration are generally known in the art and include intravenous including infusion and/or bolus injection, intracerebroventricularly, intrathecal, parenteral, mucosal, implant, intraperitoneal, oral, intradermal, transdermal (e.g., in slow release polymers), intramuscular, subcutaneous, topical, epidural, etc. routes. Other suitable routes of administration can also be used, for example, to achieve absorption through epithelial or mucocutaneous linings. Particular memory agents can also be administered by gene therapy, wherein a DNA molecule encoding a particular therapeutic protein or peptide is administered to the animal, e.g., via a vector, which causes the particular protein or peptide to be expressed and secreted at therapeutic levels in vivo.
A vector, as the term is used herein, refers to a nucleic acid vector, e.g., a DNA plasmid, virus or other suitable replicon (e.g., viral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., U.S. Pat. No. 5,801,030, the teachings of which are incorporated herein by reference.
A nucleic acid sequence encoding a protein or peptide (e.g., CREB protein, CREB analog (including CREM, ATF-1), CREB-like molecule, biologically active CREB fragment, CREB fusion protein, CREB function modulator) can be inserted into a nucleic acid vector according to methods generally known in the art (see, e.g., Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989)).
The mode of administration is preferably at the location of the target cells. In a particular embodiment, the mode of administration is to neurons.
Memory agents can be administered together with other components of biologically inactive agents, such as pharmaceutically acceptable surfactants or carriers (e.g., glycerides), excipients (e.g., lactose), stabilizers, preservatives, humectants, emollients, antioxidants, carriers, diluents and vehicles. If desired, certain sweetening, flavoring and/or coloring agents can also be added. When combined with various inactive ingredients, a memory agent may also be referred to as a pharmaceutical composition.
Memory agents can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, isotonic sodium chloride solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation can be sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences.
The dosage of memory agent administered to an animal is that amount required to effect a change in CREB-dependent gene expression, particularly in neurons. The dosage administered to an animal, including frequency of administration, will vary depending upon a variety of factors, including pharmacodynamic characteristics of the particular memory agent, mode and route of administration; size, age, sex, health, body weight and diet of the recipient; nature and extent of symptoms being treated or nature and extent of the memory being enhanced or modulated, kind of concurrent treatment, frequency of treatment, and the effect desired.
Memory agents can be administered in single or divided doses (e.g., a series of doses separated by intervals of days, weeks or months), or in a sustained release form, depending upon factors such as nature and extent of symptoms, kind of concurrent treatment and the effect desired. Other therapeutic protocols or agents can be used in conjunction with the present invention.
The teachings of all the articles, patents and patent applications cited herein are incorporated by reference in their entirety.

Example 1

A study was undertaken to examine the effects of a memory agent (HT-0712).
The study was conducted using 6 aged male macaques (age approximately 22 years at onset of the study which is equivalent to approximately 66 human years) previously trained to performed certain memory tasks. Assessments of memory performance used the CANTAB system (the Cambridge Neuropsychological Test Automated Battery) and a new task.

Discrimination Learning—General Description of a Long Term Retrieval Task (DL LTR)

In this task, animals are presented with 2 pairs of stimuli (e.g. 1 pair of shapes and 1 pair of lines) over the course of a daily test session. For each pair of stimuli, one stimulus is arbitrarily designated as the positive stimulus (i.e., touch of that stimulus is rewarded). Each pair of stimuli is presented 14 times during a daily session that consists of 28 trials. The pairs of stimuli (shapes or lines) are presented in a pseudo randomized fashion during each session. Animals have to touch the “positive” shape or line in order to receive a sugar pellet reward accompanied by a pleasant tone. Touching the alternative shape or line results in a negative tone, no sugar pellet and a time out. Animals are tested daily until they reach a performance criterion of 85% correct responses during a daily session. When this criterion is reached, animals receive no further testing for the next 7 days. On day 8 post-criterion, animals are tested again using the same pairs of stimuli that they have seen previously learned in order to assess long term retrieval of the learned discriminations.

Drug Administration

All drugs were administered orally in marshmallow while the animal was in a specially designed testing cage. Vehicle was marshmallow alone.
The treatment schedule is shown in Table 1. Treatments were administered sequentially starting with treatment 1. Each treatment (dose of HT-0712 or vehicle) was administered once per day for 4 days before behavioral training began and then continued at 1 treatment (dose of HT-0712 or vehicle) per day, delivered four hours before testing, for the duration of the training period. When each individual animal reached performance criterion of at least 85% correct responses during a daily session, training and dosing ceased for seven days. Then, on post-criterion day 8, animals were tested again on the DL LTR task (retention testing).
A second vehicle study was conducted at the end of the drug trials using the first 3 animals that completed the 100 mg/kg dosing study.

TABLE 1

Treatment Schedule

1 (N = 6)	vehicle	0	p.o.
2 (N = 6)	HT-0712	1 mg/kg	p.o.
3 (N = 6)	HT-0712	10 mg/kg	p.o.
4 (N = 6)	HT-0712	100 mg/kg	p.o.
5 (N = 3)	vehicle	0	p.o.

Analysis of Behavioral Data

The percent correct responses on the total number of daily trials were recorded as well as the number of days needed to reach the criterion (at least 85% correct responses in a single test session). The percent correct responses on the retention trial was recorded for the entire retention trial session and sub-analyzed for performance differences on trials 1-14 versus trials 15-28.
Data from vehicle trials were compared to data from drug trials using a one way ANOVA. Post hoc comparisons were made using the Bonferroni t-test.

Results

There were no noticeable behavioral or physical side effects following administration of HT-0712 at any dose given during the study. Five of the six animals used displayed improved memory performance as evidenced by a decrease in the number of days needed to reach criterion. One animal (old-9) did not show improved task performance at any dose of HT-0712 administered. Data from this non-responder were removed from the statistical analysis.

Vehicle Treatment

During the first vehicle study, all animals reached criterion over a period of 26.3±4.6 days. The mean criterion value reached was 89.3±1.8% correct responses. Mean performance at the retention trial was of 79.7±5.9% correct responses. When retention trial performance was divided between trials 1 to 14 versus trials 15 to 28, animals performed 77.5±5.1% correct responses and 82.3±7.5% correct responses, respectively.
The second (post drug study) vehicle study was performed using the first 3 animals that finished the drug trials. During this second vehicle study, animals reached criterion over a period of 20.0±4.5 days. Their mean criterion value was 86.3±1.3% correct responses. Performance at the retention trials averaged 64.0±4.0% correct responses. When retention trial performance was divided between trials 1 to 14 versus trials 15 to 28, animals performed 59.3±8.4% correct responses and 69.0±10.4% correct responses, respectively.
No statistically significant differences were found between the results of the pre-study and post-study vehicle trials (t=1.168, (P=0.363)).

Memory Agent Treatment

At the lowest dose of HT-0712 used in this study (1 mg/kg), animals reached criterion over a period of 12.0±3.6 days, at a performance level of 91.4±1.8% correct responses). The mean performance of the animals at the retention trial was of 85.8±4.6% correct responses. When retention trial performance was divided between trials 1 to 14 versus trials 15 to 28, animals performed at a level of 80.2±6.2% correct responses and 91.6±3.4% correct responses, respectively.
At the medium dose of HT-0712 used in the study (10 mg/kg), animals reached criterion (90.0±2.6% correct responses) over a period of 11.8±2.3 days. The mean performance of these animals at retention testing was of 79.2±3.5% correct responses. When retention test performance was divided between trials 1 to 14 versus trials 15 to 28, animals performed at a level of 71.4±3.4% correct and 87.2±4.8% correct, respectively.
At the highest dose of HT-0712 used (100 mg/kg), animals reached criterion (86.4±0.7% correct responses) over a period of 9.0±2.8 days. The mean performance of these animals at retention testing was of 65.0±3.5% correct responses. When retention test performance was divided between trials 1 to 14 versus trials 15 to 28, animals performed at a level of 58.4±4.1% correct and 71.4±6.1% correct, respectively.
The effect of treatment on days to criterion was significant (repeated measures ANOVA, F(4,3)=6.327, p=0.008). Post hoc comparisons using the Bonferroni t-test showed a significant difference between vehicle and each dose of HT-0712 (p<0.050 for each comparison).
There was no significant effect of treatment on the level of performance attained on the criterion day (F(4,3)=1.408, p=0.289).
There was no significant effect of treatment on the level of performance attained at the retention test (F(4,3)=3.199, p=0.062). Analysis of treatment effect on retention trial performance at trials 1-14 and trials 15-28, also failed to reach statistical significance (p=0.058 and p=0.167, respectively).
This study was conducted using a new task, the Discrimination Learning—Long Term Retrieval task (DL LTR), that was designed to assess the effects of an experimental memory agent on learning and retention in aged non human primates.
The results of the vehicle control studies indicate that normal aged animals require a long time (ex. >20 days) to reach an 85% correct response criterion on this test. Additionally, the post-drug vehicle test results indicate that the increase in performance seen with HT-0712 was not due to an increase in the animal's proficiency in the test or a practice effect as the second vehicle control test results were not significantly different from the results of the first vehicle study.
Five of the six animals tested showed improved performance following administration of HT-0712. The improvement was expressed as a significant decrease in the number of days to reach criterion, showing improved learning abilities when treated with HT-0712.
The response of the animals could be separated into 2 general groups. In the first group (animals old-5 and old-8), the best dose for improving performance was the 1 mg/kg dose of HT-0712. These animals showed a decrement in performance as the dose administered increased from 1 to 10 to 100 mg/kg. That is, with increasing doses of HT-0712, it took longer for the animals to reach criterion (i.e., 4, 5, and 10 days to criterion (old-5) and 3, 12, and 18 days to criterion for (old-8) after administration of 1, 10, or 100 mg/kg, respectively). The second group of animals (old-1, old-2 and old-7) showed a more typical dose-response effect in which performance improved with increasing doses (21, 15, and 3 days to criterion; 17, 18, and 11 days to criterion, and 15, 9, and 3 days to criterion for old-1, old2 and old-7 respectively with doses of 1, 10, and 100 mg/kg).
The present animals were previously tested and assessed for cognitive deficits using both delayed matching to sample (DMTS) and paired associative learning (PAL) tasks. These animals were ranked accordingly to their level of performance on these tasks (i.e., animals were ranked per task from 1 to 6, 1 being the best performer and 6 the worst (see Table 10). This ranking showed that the animal that did not respond to HT-0712 was ranked 1 on performance of the PAL and 2 on performance of the DMTS. This same animal also had the best performance on the first vehicle study performed on the current project.
In summary, the results of this study show that HT-0712 significantly improves the learning ability and long term memory of normal aged non-human primates using particular systems and methods of the present invention.

Tables

TABLE 2

Descriptive statistics for the entire study group, including NHP old-9

	Criterion	Criterion
Mean	Deviation	Error

vehicle day to C	26.333	11.237	4.587
vehicle C value	89.333	4.274	1.745
vehicle retention	79.667	14.459	5.903
test
vehicle RT 1-14	77.500	12.486	5.097
vehicle RT 15-28	82.333	18.468	7.540
vehicle2 day to C	20.000	7.810	4.509
vehicle2 C value	86.333	2.309	1.333
vehicle retention	64.000	7.000	4.041
test
vehicle2 RT 1-14	59.333	14.572	8.413
vehicle2 RT 15-28	69.000	18.083	10.440
1 mg day to C	12.667	7.394	3.018
1 mg C value	91.000	3.633	1.483
1 mg retention test	82.833	11.788	4.813
1 mg RT 1-14	77.500	14.068	5.743
1 mg RT 15-28	88.167	10.852	4.430
10 mg day to C	14.000	7.043	2.875
10 mg C value	89.333	5.428	2.216
10 mg retention	80.333	7.528	3.073
test
10 mg RT 1-14	73.833	8.976	3.664
10 mg RT 15-28	87.000	9.633	3.933
100 mg day to C	10.333	6.501	2.654
100 mg C value	86.333	1.366	0.558
100 mg retention	66.000	7.430	3.033
test
100 mg RT 1-14	59.333	8.477	3.461
100 mg RT 15-28	72.667	12.580	5.136

Legends:
C: criterion;
RT: retention test,
RT 1-14 Retention test trials 1 to 14;
RT 15-28: retention test trials 15 to 28

TABLE 3

Descriptive statistics for the entire study group, excluding NHP old-9

	Criterion	Criterion
Mean	Deviation	Error

Vehicle1 day to C	29.000	10.223	4.572
Vehicle1 C value	88.600	4.336	1.939
Vehicle1 retention	79.200	16.115	7.207
test
Vehicle1 RT 1-14	77.200	13.936	6.232
Vehicle1 RT 15-28	81.600	20.550	9.190
vehicle2 day to C	22.000	9.899	7.000
vehicle2 C value	85.000	0.000	0.000
Vehicle2 retention	64.000	9.899	7.000
test
vehicle2 RT 1-14	67.500	4.950	3.500
vehicle2 RT 15-28	60.500	14.849	10.500
1 mg day to C	12.000	8.062	3.606
1 mg C value	91.400	3.912	1.749
1 mg retention test	85.800	10.378	4.641
1 mg RT 1-14	80.200	13.882	6.208
1 mg RT 15-28	91.600	7.668	3.429
10 mg day to C	11.800	5.070	2.267
10 mg C value	90.000	5.788	2.588
10 mg retention	79.200	7.823	3.499
test
10 mg RT 1-14	71.400	7.503	3.356
10 mg RT 15-28	87.200	10.756	4.810
100 mg day to C	9.000	6.285	2.811
100 mg C value	86.400	1.517	0.678
100 mg retention	65.000	7.842	3.507
test
100 mg RT 1-14	58.400	9.127	4.082
100 mg RT 15-28	71.400	13.631	6.096

Legends:
C: criterion;
RT: retention test,
RT 1-14 Retention test trials 1 to 14;
RT 15-28: retention test trials 15 to 28

Raw data per treatment

TABLE 4

Vehicle 1

			RT	RT trials	RT trials
Treatment	Days to C	C value	overall	1-14	15-28

Old-1	Vehicle 1	43	86.00	54.00	57.00	50.00
Old-2	Vehicle 1	18	86.00	89.00	79.00	100.00
Old-5	Vehicle 1	36	96.00	96.00	93.00	100.00
Old-7	Vehicle 1	25	86.00	82.00	86.00	79.00
Old-8	Vehicle 1	23	89.00	75.00	71.00	79.00
Old-9	Vehicle 1	13	93.00	82.00	79.00	86.00

TABLE 5

1 mg/kg HT-0712

			RT	RT trials	RT trials
Treatment	Days to C	C value	overall	1-14	15-28

Old-1	1 mg/kg	21	86.00	93.00	93.00	93.00
Old-2	1 mg/kg	17	93.00	86.00	79.00	93.00
Old-5	1 mg/kg	4	89.00	93.00	86.00	100.00
Old-7	1 mg/kg	15	93.00	68.00	57.00	79.00
Old-8	1 mg/kg	3	96.00	89.00	86.00	93.00
Old-9	1 mg/kg	16	89.00	68.00	64.00	71.00

TABLE 6

10 mg/kg HT-0712

			RT	RT trials	RT trials
Treatment	Days to C	C value	overall	1-14	15-28

Old-1	10 mg/kg	15	86.00	75.00	64.00	86.00
Old-2	10 mg/kg	18	86.00	71.00	71.00	71.00
Old-5	10 mg/kg	5	100.00	86.00	79.00	93.00
Old-7	10 mg/kg	9	89.00	75.00	64.00	86.00
Old-8	10 mg/kg	12	89.00	89.00	79.00	100.00
Old-9	10 mg/kg	25	86.00	86.00	86.00	86.00

TABLE 7

100 mg/kg HT-0712

			RT	RT trials	RT trials
Treatment	Days to C	C value	overall	1-14	15-28

Old-1	100 mg/kg	3	89.00	57.00	50.00	64.00
Old-2	100 mg/kg	11	86.00	68.00	50.00	86.00
Old-5	100 mg/kg	10	86.00	57.00	57.00	57.00
Old-7	100 mg/kg	3	86.00	68.00	71.00	64.00
Old-8	100 mg/kg	18	85.00	75.00	64.00	86.00
Old-9	100 mg/kg	17	86.00	71.00	64.00	79.00

TABLE 8

Vehicle 2

			RT	RT trials	RT trials
Treatment	Days to C	C value	overall	1-14	15-28

Old-1	Vehicle 2	15	85.00	71.00	71.00	71.00
Old-5	Vehicle 2	29	85.00	57.00	64.00	50.00
Old-9	Vehicle 2	16	89.00	64.00	43.00	86.00

Legends:
C: criterion;
RT: retention test,
RT 1-14 Retention test trials 1 to 14;
RT 15-28: retention test trials 15 to 28

TABLE 9

Description of animals used in the study

ID	Weight at start of study	Estimated age	Sex

Old-1	9.9 kg	22 years old	Male
Old-2	9.65 kg	22 years old	Male
Old-5	8.4 kg	22 years old	Male
Old-7	10.6 kg	22 years old	Male
Old-8	9.8 kg	22 years old	Male
Old-9	11.15 kg	22 years old	Male

TABLE 10

Ranking of animals used in the study on PAL and DMTS

old-1 old-2 old-5 old-7 old-8 old-9

PAL 4 5 6 2 3 1

DMTS 6 1 5 4 3 2

mean rank 5 3 5.5 3 3 1.5

Rankings were calculated for PAL and DMTS task as follows:
1. PAL: Mean number of trials needed to complete the most difficult level of the task (3 stimuli/3 locations).
2. DMTS: Mean number of correct response at the longer delay.
Animals were ranked per task from 1 to 6, 1 being the best performer and 6 the worst.

Example 2

Single-Blind Study in Healthy, Elderly, Human Subjects

Study Design

The study using the general protocol and drug administration as set forth in Example 1 can be performed using humans. As described below, a long term retrieval task can utilize stimuli in the form of face—name and/or occupation pairings. The human subjects can be shown a picture of a face along with the name and/or occupation of a person. An occupation may be listed for each name-face pair. During encoding, the human subject is asked to decide if the name fits with the occupation. During the test, the human subject is shown intact, rearranged, and novel face-name pairs (presented without the occupation) and asked to decide if the face and name pairings are intact, rearranged, or novel. Using this testing, human subjects can then be evaluated in a manner as set forth in Example 1.
In one embodiment, a single site, double-blind, three-way cross-over study was performed that tested the following three dosing regimens: (1) HT-0712 45 mg po for 7 days; (2) HT-0712 45 mg po plus warfarin 2.5 mg po for 7 days; and (3) Warfarin 2.5 mg po for 7 days. There was a seven-day washout periods between dosing periods. Subjects remained in-house for three (72 hours) of the seven days washout days, for study related procedures. Subjects were furloughed from the clinic for four of the seven washout days. With regard to subject selection criteria, there were no inclusion and/or exclusion criteria for the paired associate test.

Study Schedule

Group I subjects received 45 mg doses of HT-0712 on Days 1,2,3,4,5,6, and 7; 45 mg doses of HT-0712 plus 2.5 mg doses of warfarin on Days, 15, 16, 17, 18, 19, 20, and 21; and 2.5 mg doses of warfarin on Days 29, 30, 31, 32, 33, 34, and 35. Group II subjects received 45 mg doses of HT-0712 plus 2.5 mg doses of warfarin on Days 1,2,3,4,5,6, and 7; 2.5 mg doses of warfarin on Days 15, 16, 17, 18, 19, 20, and 21; and 45 mg doses of HT-0712 on Days 29, 30, 31, 32, 33, 34, and 35. Group III subjects received 2.5 mg doses of warfarin on Days 1,2,3,4,5,6, and 7; 45 mg doses of HT-0712 on Days 15, 16, 17, 18, 19, 20, and 21; and 45 mg doses of HT-0712 plus 2.5 mg doses of warfarin on Days 29, 30, 31, 32, 33, 34, and 35.

TABLE 11

Unblinded Subject Codes

Group

1	Group 2	Group 3

002	001	003
005	004	006
008	007	009
012	011	010
015	013	014
016	018	017
019	020	021

Paired Associates Testing

Materials: Materials consisted of 21 Lenovo notebook computers. Each notebook was configured to eliminate all auto-configuration software and reduce settings which might interfere with the internal clock settings and E-Run software. E-Run (v1.2.1.68) was installed on all the notebook computers to administer the paired associate test. 120 grayscale faces (male and female) were paired with a two-word professions, such as “fire fighter.”Grayscale face images were drawn from a face database from Lund University Cognitive Science Lab. Only two-word or compound-word professions were used. The 120 face-name pairs were divided into three groups of 40. During each round the same 40 face-name pairs were used throughout the round.
Procedure—Timing of Administration: Memory testing commenced approximately 6 hours post-drug administration. All memory testing was completed prior to daily blood samples being taken. The training was performed in three Rounds (or Segments) consisting of 14 days as shown in Table 12. The testing was performed over 42 days. In a given round on day 1, Subjects were familiarized with the computers and trained how to use the test software. On day 2 subjects were given an encoding and retrieval session. On days 3-7, subjects were tested daily for recall. On day 10 subjects were tested on half the face-name pairs, and on day 14, subjects were tested on the remaining half of the face-name pairs followed by a complete test of all face-name pairs used in that round.

TABLE 12

Training and Testing

Round

1 Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14

Procedure	Training	Encoding	Testing	Testing	Testing	Testing	Testing			Testing				Testing
# of Pairs		40 Pairs	40	40	40	40	40			Pairs				Part I:
			Pairs	Pairs	Pairs	Pairs	Pairs			1-20				Pairs
														21-40;
														Part II:
														Repeat
														all 40
														Pairs

Testing Procedure Familiarization: On day 1, subjects were familiarized with the computers and trained how to use the test software by receiving a mock encoding and retrieval session consisting of nine face-name pairs. These pairs were never observed again.
Encoding and Retrieval: On Day 2, each participant received a single encoding and retrieval session:
Encoding: During the encoding session, participants see 40 faces paired with professions one at a time on a computer screen. For instance they see a face, and to the right, the profession “ice skater.” Their task for each face-profession pair is to form a vivid mental image in which the person pictured is carrying out the profession appearing to the right. Thus, for a face paired with ice skater, they might imagine that person doing a difficult jump and falling, or imagine them very happy because they've won the gold medal. After 5 seconds of viewing the face profession pair, they were prompted to rate how easy it was to form the mental image on a scale of 1-3: 1 if it was easy, 2 if it was difficult, 3 if they were unsuccessful at creating a mental image. After 2 seconds, the next face profession pair was displayed.
Retrieval: In this part of the experiment, participants were administered a cued-recall test for the 40 face-profession pairs they had seen in during Encoding. During the retrieval test subjects were presented face without the associated profession. Next to the face there was a box for them to type in the profession that was paired with that face during encoding. Once the participant types a response, they received feedback as to whether the profession they typed was correct or incorrect. If the response was correct, “Correct!” appeared on the screen with the appropriate face and profession pairing, followed by the next stimulus pair. If it was incorrect, the correct face and profession pairing was presented as feedback on the screen for three seconds, prior to the presentation of the next stimulus pair. The session ended once all forty face-profession pairs were presented. This retrieval session was presented once, each day 3-7, of the study.
Long-term memory assessment: Three days after each final drug dose and prior to being discharged (day 10, 24, and 38) participants were administered a cued-recall test consisting of 20 face-profession pairs to measure long term stability of the face-profession memories. Seven days after each drug treatment (day 14 and 28, and 42), participants were administered the remaining 20 face-profession pairs which were not previously tested. On days 14 and 28, and 42, a minute rest period followed the presentation of the 20 face-profession pairs. Following this one minute of rest, participants were administered the complete set of 40 face-profession pairs.
Results: Data reflecting participant recall was collected and graphed as shown in FIG. 5. These data show that long-lasting memory of a paired-associate task can be established with sufficient daily repetitions of training.
Blinding Procedure: The study was a double-blind placebo controlled trial. The pharmacist had responsibility for maintaining subject treatment codes.
Thus, it is seen that various methods are provided. One skilled in the art will appreciate that the present invention can be practiced by other than the various embodiments and preferred embodiments, which are presented in this description for purposes of illustration and not of limitation, and the present invention is limited only by the claims that follow. It is noted that equivalents for the particular embodiments discussed in this description may practice the invention as well.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not of limitation. Likewise, the various figures may depict an example configuration for the invention, which is done to aid in understanding the features and functionality that may be included in the invention. The invention is not restricted to the illustrated example configurations, but the desired features may be implemented using a variety of alternative configurations. Indeed, it will be apparent to one of skill in the art how alternative functional, logical or physical configurations may be implemented to implement the desired features of the present invention. Additionally, with regard to method claims, the order in which the steps are presented herein shall not mandate that various embodiments be implemented to perform the recited functionality in the same order unless the context dictates otherwise.
Although the invention is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features, aspects and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described, but instead may be applied, alone or in various combinations, to one or more of the other embodiments of the invention, whether or not such embodiments are described and whether or not such features are presented as being a part of a described embodiment. Thus the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments.
Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term “including” should be read as meaning “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof, the terms “a” or “an” should be read as meaning “at least one,” “one or more” or the like; and adjectives such as “conventional,” “traditional,” “normal,” “criterion,” “known” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or criterion technologies that may be available or known now or at any time in the future. Likewise, where this document refers to technologies that would be apparent or known to one of ordinary skill in the art, such technologies encompass those apparent or known to the skilled artisan now or at any time in the future.
A group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless expressly stated otherwise. Furthermore, although items, elements or components of the invention may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated.
The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and their various alternatives may be implemented without confinement to the illustrated examples.

Claims

1. A method comprising the steps of:

assessing the effectiveness of a memory agent administered to a subject, wherein said assessing comprises presenting to said subject sets of stimuli; and

evaluating said subject's response to said stimuli.

2. The method of claim 1, wherein said stimuli affect a CREB pathway.

3. The method of claim 2, wherein said stimuli affect the subject's long term memory.

4. The method of claim 1, wherein said assessing further comprises selecting a performance criterion and evaluating the number of days of training required for the subject to reach the performance criterion.

5. The method of claim 1 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

6. The method of claim 5 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

7. The method of claim 6 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

8. The method of claim 1 wherein said subject is an animal.

9. The method of claim 8 wherein said animal is a mammal.

10. The method of claim 9 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

11. The method of claim 10 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

12. The method of claim 11 wherein said primate is a human.

13. The method of claim 1 wherein said subject is a plurality of subjects.

14. The method of claim 13 wherein said plurality of subjects comprises a control group and an experimental group.

15. The method of claim 14 wherein said assessing comprises comparing said control group to said experimental group.

16. The method of claim 1, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

wherein each of Y₁, Y₂, Y₃, and Y₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl or C₅-C₇cycloalkenyl; —F, —Cl, —Br, or —I; —NO₂; —N₃; —CN; —OR₄, —SR₄, —OCOR₄, —COR₄, —NCOR₄, —N(R₄)₂, —CON(R₄)₂, or —COOR₄; aryl or heteroaryl; or any two of Y₁, Y₂, Y₃and Y₄present on adjacent carbon atoms can constitute a methylenedioxy group; wherein each R₄is independently —H; straight chained or branched C₁-C₇alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C₂-C₇alkenyl or alkynyl; C₃-C₇cycloalkyl, C₅-C₇cycloalkenyl, aryl or aryl(C₁-C₆)alkyl; wherein A is A′, straight chained or branched C₁-C₇alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl or heteroaryl(C₁-C₆)alkyl; wherein A′ is

wherein R₁and R₂are each independently H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, or —CN; wherein R₃is H, straight chained or branched C₁-C₇alkyl, —F, —Cl, —Br, —I, —NO₂, —CN, —OR₆, aryl or heteroaryl; wherein R₅is straight chained or branched C₁-C₇alkyl, —N(R₄)₂, —OR₄or aryl; wherein R₆is straight chained or branched C₁-C₇alkyl or aryl; wherein B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-napthyridinyl, pteridinyl, or phthalimidyl; provided however, that the carbon atom or carbon atoms ortho to the nitrogen atom of the imine bond may only be substituted with one or more of the following —F, —Cl, —Br, —I, —CN, methyl, ethyl or methoxy; wherein n is an integer from 1 to 4 inclusive; wherein aryl is phenyl or napthyl, including phenyl and napthyl substituted with one or more of the following: —F, —Cl, —Br, —I, —NO₂, —CN, straight chained or branched C₁-C₇alkyl, straight chained or branched C₁-C₇monofluoroalkyl, straight chained or branched C₁-C₇polyfluoroalkyl, straight chained or branched C₂-C₇alkenyl, straight chained or branched C₂-C₇alkynyl, C₃-C₇cycloalkyl, C₁-C₇monofluorocycloalkyl, C₃-C₇polyfluorocycloalkyl, C₅-C₇cycloalkenyl, —OR₄, SR₄, —OCOR₄, —COR₄, —NCOR₄, —CO₂R₄, —CON(R₄)₂or (CH₂)_n—O—(CH₂)_mCH₃; or a pharmaceutically acceptable salt thereof.

17. The method of claim 1, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

wherein Me is methyl, cPent is cyclopentyl and the 3 and 5 carbons are in the S configuration.

18. A method comprising the steps of assessing the effectiveness of a memory agent administered to a subject, wherein said assessing comprises selecting a performance criterion, training the subject by presenting the subject with sets of stimuli, evaluating the subject's response to the stimuli to determine whether the subject has reached the performance criterion, and determining the number of days of training required to reach the performance criterion.

19. The method of claim 18 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

20. The method of claim 19 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

21. The method of claim 20 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

22. The method of claim 18 wherein said stimuli affect a CREB pathway.

23. The method of claim 18 wherein said stimuli affect the subject's long term memory.

24. The method of claim 18 wherein the performance criterion is a percentage representing the quality of the subject's response to the stimuli.

25. The method of claim 18, wherein the performance criteria represents the subject's acquisition of long term memory.

26. The method of claim 18 wherein said subject is an animal.

27. The method of claim 26 wherein said animal is a mammal.

28. The method of claim 27 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

29. The method of claim 28 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

30. The method of claim 29 wherein said primate is a human.

31. The method of claim 18 wherein said subject is a plurality of subjects.

32. The method of claim 31 wherein said plurality of subjects comprises a control group and an experimental group.

33. The method of claim 32 wherein said assessing comprises comparing said control group to said experimental group.

34. The method of claim 18, wherein said, memory agent is one or more selected from the group consisting of a compound having the structure:

35. The method of claim 18, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

36. A method comprising the steps of assessing the effectiveness of a memory agent administered to a subject, wherein said assessing comprises selecting a performance criterion, training the subject by presenting the subject with sets of stimuli, evaluating the subject's response to said stimuli to determine whether the subject has reached the performance criterion, repeating said presenting and evaluating until the subject reaches the performance criterion, and determining the number of days of training required for the subject to reach the performance criterion.

37. The method of claim 36, wherein said stimuli affect a CREB pathway.

38. The method of claim 36, wherein said stimuli affect the subject's long term memory.

39. The method of claim 36 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

40. The method of claim 39 wherein each of said plurality of pairs of stimuli comprises a element that has an identifiable association with the other element of said pair.

41. The method of claim 40 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

42. The method of claim 36 wherein said subject is an animal.

43. The method of claim 42 wherein said animal is a mammal.

44. The method of claim 43 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

45. The method of claim 44 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

46. The method of claim 45 wherein said primate is a human.

47. The method of claim 36 wherein said subject is a plurality of subjects.

48. The method of claim 47 wherein said plurality of subjects comprises a control group and an experimental group.

49. The method of claim 48 wherein said assessing comprises comparing said control group to said experimental group.

50. The method of claim 36, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

51. The method of claim 36 wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

52. A method comprising selecting a memory agent as a drug candidate, wherein said selecting comprises the steps of assessing said memory agent by administering said memory agent to a subject; and presenting to said subject sets of stimuli and evaluating said subject's response to said stimuli.

53. The method of claim 52 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

54. The method of claim 53 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

55. The method of claim 54 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

56. The method of claim 52 wherein said stimuli affect a CREB pathway.

57. The method of claim 52 wherein said stimuli affect the subject's long term memory.

58. The method of claim 52, wherein said assessing further comprises selecting a performance criterion, and said evaluating comprises determining the quantity of stimuli required for the subject to reach the performance criterion.

59. The method of claim 58, wherein the performance criterion is a percentage representing the quality of the subject's response to the stimuli.

60. The method of 59, wherein the performance criteria represents the subject's acquisition of long term memory.

61. The method of claim 52 wherein said subject is an animal.

62. The method of claim 61 wherein said animal is a mammal.

63. The method of claim 62 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

64. The method of claim 63 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

65. The method of claim 64 wherein said primate is a human.

66. The method of claim 52 wherein said subject is a plurality of subjects.

67. The method of claim 66 wherein said plurality of subjects comprises a control group and an experimental group.

68. The method of claim 67 wherein said assessing comprises comparing said control group to said experimental group.

69. The method of claim 52, wherein said, memory agent is one or more selected from the group consisting of a compound having the structure:

70. The method of claim 52, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

71. A method comprising:

testing a memory agent as a long term memory enhancer, wherein said testing comprises the steps of:

administering said memory agent to a subject;

presenting to said subject sets of stimuli; and

evaluating said subject's response to said stimuli.

72. The method of claim 71 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

73. The method of claim 72 wherein each of said plurality of pairs of stimuli comprises a element that has an identifiable association with the other element of said pair.

74. The method of claim 73 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

75. The method of claim 71 wherein the second subject is an animal.

76. The method of claim 75, wherein the animal is a mammal.

77. The method of claim 76 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

78. The method of claim 77, wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

79. The method of claim 78 wherein said primate is a human.

80. The method of claim 71 wherein said subject is a plurality of subjects.

81. The method of claim 80 wherein said plurality of subjects comprises a control group and an experimental group.

82. The method of claim 81 wherein said assessing comprises comparing said control group to said experimental group.

83. The method of claim 71, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

84. The method of claim 71, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

85. A system comprising: sets of stimuli, wherein said sets of stimuli are configured to permit evaluation of a subject's response to said sets of stimuli and thereby assess the effectiveness of a memory agent administered to said subject.

86. The system of claim 85 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

87. The system of claim 86 wherein each of said plurality of pairs of stimuli comprises a positive element or a negative element.

88. The system of claim 86 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

89. The system of claim 88 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

90. The system of claim 85 wherein said subject is an animal.

91. The system of claim 90 wherein said animal is a mammal.

92. The system of claim 91 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

93. The method of claim 92 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

94. The method of claim 93 wherein said primate is a human.

95. The system of claim 85 wherein said subject is a plurality of subjects.

96. The system of claim 95 wherein said plurality of subjects comprises a control group and an experimental group.

97. The system of claim 96 wherein said assessing comprises comparing said control group to said experimental group.

98. The system of claim 85, wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

99. The system of claim 85 wherein said memory agent is one or more selected from the group consisting of a compound having the structure:

100. A method comprising the steps of:

assessing the effectiveness of a training protocol administered to a subject, wherein said assessing comprises presenting to said subject sets of stimuli and evaluating said subject's response to said stimuli.

101. A method comprising:

testing a training protocol as a long term memory enhancer, wherein said testing comprises the steps of:

administering a training protocol to a subject; and

presenting to said subject sets of stimuli and evaluating said subject's response to said stimuli.

102. A system comprising: sets of stimuli, wherein said sets of stimuli are configured to permit evaluation of a subject's response to said sets of stimuli and thereby assess the effectiveness of a training protocol administered to said subject.

103. The system of claim 102 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

104. The system of claim 103 wherein each of said plurality of pairs of stimuli comprises a positive element or a negative element.

105. The system of claim 103 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

106. The system of claim 105 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

107. A method comprising the steps of:

assessing the effectiveness of a memory impairer administered to a subject, wherein said assessing comprises presenting to said subject sets of stimuli and evaluating said subject's response to said stimuli.

108. The method of claim 107 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

109. The method of claim 108 wherein each of said plurality of pairs of stimuli comprises a positive element or a negative element.

110. The method of claim 108 wherein each of said plurality of pairs of stimuli comprises a element that has an identifiable association with the other element of said pair.

111. The method of claim 110 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

112. The method of claim 107 wherein said subject is an animal.

113. The method of claim 112 wherein the animal is a mammal

114. The method of claim 113 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

115. The method of claim 114 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

116. The method of claim 115 wherein said primate is a human.

117. The method of claim 107 wherein said subject is a plurality of subjects.

118. The method of claim 117 wherein said plurality of subjects comprises a control group and an experimental group.

119. The method of claim 118 wherein said assessing comprises comparing said control group to said experimental group.

120. A method comprising:

selecting a memory impairer as a drug candidate, wherein said selecting comprises the steps of:

assessing said memory impairer by

administering said memory impairer to a subject; and

presenting to said subject sets of stimuli and

evaluating said subject's response to said stimuli.

121. The method of claim 120 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

122. The method of claim 121 wherein each of said plurality of pairs of stimuli comprises a positive element or a negative element.

123. The method of claim 121 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

124. The method of claim 123 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

125. The method of claim 120 wherein the subject is an animal.

126. The method of claim 125, wherein said animal is a mammal.

127. The method of claim 126 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

128. The method of claim 127 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

129. The method of claim 128 wherein said primate is a human.

130. The method of claim 120 wherein said subject is a plurality of subjects.

131. The method of claim 130 wherein said plurality of subjects comprises a control group and an experimental group.

132. The method of claim 131 wherein said assessing comprises comparing said control group to said experimental group.

133. A system comprising: sets of stimuli, wherein said sets of stimuli are configured to permit evaluation of a subject's response to said sets of stimuli and thereby assess the effectiveness of a memory impairer administered to said subject.

134. The system of claim 133 wherein said sets of stimuli comprise a plurality of pairs of stimuli.

135. The system of claim 134 wherein each of said plurality of pairs of stimuli comprises a positive element or a negative element.

136. The system of claim 134 wherein each of said plurality of pairs of stimuli comprises an element that has an identifiable association with the other element of said pair.

137. The system of claim 136 wherein said identifiable association is selected from the group consisting of a face—name association and a word—word association.

138. The system of claim 133 wherein the subject is an animal.

139. The system of claim 138, wherein said animal is a mammal.

140. The system of claim 139 wherein said mammal is selected from the group consisting of a primate, a mouse and a rat.

141. The system of claim 140 wherein said primate is selected from the group consisting of a human, a monkey, a lemur, a macaque and an ape.

142. The system of claim 141 wherein said primate is a human.

143. The system of claim 133 wherein said subject is a plurality of subjects.

144. The system of claim 143 wherein said plurality of subjects comprises a control group and an experimental group.

145. The system of claim 144 wherein said assessing comprises comparing said control group to said experimental group.