Patent Application
20090221610
The present invention relates to the use of inhibitors or blockers of Ih (hyperpolarization-activated cationic current) channels in the treatment of cognitive disorders.
Hyperpolarization-activated cationic current channels (Ih) were initially identified in cardiac myocetes and photoreceptors. Brown, et al., Nature, 280, 235-236 (1979), Brown and DiFrancesco, J. Physiol., and Bader, et al., J. Physiol., 2961-216 (1979). The currents are characterized by permeability to both K+ and Na+, and modulation by direct binding of cAMP, which is needed for channel opening and shifts activation to more positive channels. DiFrancesco and Tortora, Nature, 351, 145-147 (1991).
The prefrontal cortex (PFC) regulates human behavior using working memory, inhibiting inappropriate impulses and reducing distractibility (Goldman-Rakic, Phil Trans R Soc London, 351: 1445-1453, 1996; Robbins, Phil Trans R Soc London, 351: 1463-1471, 1996). The cardinal symptoms of attention-deficit/hyperactivity disorder (ADHD)—poor attention regulation, impulsivity, and hyperactivity—may all arise from weakened PFC regulation of behavior and thought. The PFC has massive connections to motor and sensory cortices and to subcortical structures such as the caudate and cerebellum. These circuits regulate attention and action, inhibiting inappropriate thoughts and behaviors and coordinating goal-directed actions.
Neuropsychological studies first identified marked impairments in ADHD patients performing tasks requiring PFC function, and imaging studies confirmed both structural and functional insufficiencies in PFC circuits. Attention-deficit/hyperactivity disorder patients also show evidence of genetic alterations, including genes related to catecholamine neurotransmission. See, Arnsten and Li, Biol. Psychiatry, 57, 1377-1384 (2005). PFC deficits have also been observed in a number of other neuropsychiatric disorders (e.g. schizophrenia, bipolar disorder, Posttraumatic Stress Disorder, Anxiety disorders, Tourettes Syndrome), in normal aging, in neurodegenerative disorders such as Alzheimer's and Parkinson's Disease, and following traumatic brain injury to the PFC.
The PFC subserves working memory. Working memory is the ability to bring to mind an event from long term storage, or keep in mind an event that has just occurred, and retain this information in a temporary buffer in order to guide behavior and thought. Given the short term nature of working memory, it cannot involve architectural changes such as structural changes in synapses, as is thought to occur with long term memory consolidation. Rather, working memory is thought to arise from a network of PFC neurons with shared properties, engaged in recurrent excitation. The spatial working memory characteristics of neurons in the primate PFC (area 46) have been well characterized in monkeys performing spatial working memory tasks that require the animal to remember a visually cued spatial location over a brief delay period. The cued position continuously changes, requiring constant updating of spatial working memory. Goldman-Rakic, Neuron 14, 477-485 (1995) identified PFC microcircuits with spatially tuned mnemonic activity during the delay period: pyramidal neurons with similar spatial tuning properties mutually exciting each other, and those with dissimilar spatial properties inhibiting each other via GABAergic interneurons. The horizontal connectivity of layer III pyramidal cells is thought to provide the anatomical basis for PFC microcircuits (Kritzer, J Comp Neurol., 359:131-143, 1995 and Goldman-Rakic, Neuron 14, 477-485 (1995)). These connections allow PFC neurons to continue firing during the delay period when no stimulus is available in the environment, maintaining representations over time even in the presence of distracting stimuli (Miller, et al., J. Neurosci. 16:5154-5167, 1996). This is a fragile process that is highly dependent on the correct neurochemical environment.
Catecholamines have an essential influence on PFC spatial working memory functions. Extensive depletion of catecholamines in PFC (area 46) is as devastating as removing the cortex itself (Brozoski, et al., Science, 205:929-931, 1979). Although early work focused on dopamine actions, it is now known that norepinephrine (NE) has a critical influence via post-synaptic a2A-adrenoceptors (Arnsten and Goldman-Rakic, Science, 230:1273-1276, 1985; Franowicz, et al., J. Neurosci, 22:8771-8777, 2002), and that blockade of these receptors in PFC profoundly impairs spatial working memory (Li and Mei, Behav Neural Biol, 62:134-139, 1994) and erodes delay-related firing (Sawaguchi, J Neurophysiology, 80:2200-2205, 1998; and Li, et al, Neuropsychopharmacol. 21:601-610, 1999). Conversely, stimulation of post-synaptic a2A-adrenoceptors strengthens PFC cognitive functions in mice (Franowicz, et al., J Neurosci, 22:8771-8777, 2002), rats (Tanila, et al., Brain Res Bull 40:117-119, 1996), monkeys (Arnsten and Goldman-Rakic, Science, 230:1273-1276, 1985; Arnsten, et al, J Neurosci, 8:4287-4298, 1988; Cai, et al., Brain Res., 614:191-196, 1993; Rama, et al., Pharmacol Biochem Behav., 54:1-7, 1996; Mao, et al., Biol Psychiatry, 46:1259-1265 1999; Wang et al., Brain Res. 1024:176-182, 2004) and humans (Jakala, et al., Neuropsychopharmacol, 20:119-130, 1999b; Jakala, et al., Neuropsychopharmacology, 20:460-470, 1999a). On the basis of this research in animals, the a2A-adrenoceptor agonist guanfacine is currently in use for treating PFC cognitive deficits in patients with Attention Deficit Hyperactivity Disorder (Hunt, et al., Amer Acad Child Adoles Psychiatry, 34:50-54, 1995; Scahill, et al., Amer J Psychiatry, 158:1067-1074, 2001; Taylor and Russo, J Clin Psychopharm 21:223-228, 2001), Tourettes Syndrome (Scahill, et al., Amer J Psychiahy, 158:1067-1074, 2001) and mild traumatic brain injury involving the PFC (McAllister, et al., Brain Inj., 18:331-350, 2004). Guanfacine is also being tested in patients with schizophrenia, pervasive development disorders and post-traumatic stress disorder. However, the molecular events underlying these therapeutic effects have not been known.
a2A-Adrenoceptor stimulation improves working memory via suppression of cAMP intracellular signaling, consistent with a2A-adrenoceptor coupling with Gi proteins. In contrast to long term memory consolidation processes which are strengthened by cAMP signaling, working memory performance is impaired by infusions of the cAMP analog, Sp-cAMPS, into the rat PFC (Taylor, et al., J Neuroscience (Online) 19:RC23, 1999). Similarly, the working memory performance of aged monkeys is impaired by systemic administration of the PDE4 inhibitor rolipram, which increases endogenous levels of cAMP (Ramos et al., Neuron, 40:835-845, 2003, Ramos et al., Learning and Memory, zxc: zxc, 2006). Recently, the enhancing effects of guanfacine have been blocked by co-infusion of Sp-cAMPS into the rat PFC (Ramos et al, Learning and Memory, zxc: sac, 2006), using low doses of Sp-cAMPS that had no effect on their own (Ramos et al, Neuron, 40:835-845, 2003). Conversely, inhibition of cAMP actions with Rp-cAMPS infusions into rat PFC improved working memory performance similar to findings with guanfacine (Ramos, et al., Neuron, 40:835-845, 2003).
cAMP signaling impairs PFC cognitive operations at the cellular level as well. Recent evidence from in vitro recordings of neurons from PFC slices indicate that cAMP may reduce network connectivity by opening Ih channels, thus lowering membrane resistance and functionally weakening cortical connectivity (David McCormick, unpublished data). 1 h channels have been shown to have important effects on dendritic integration in hippocampus, where they are localized on the distal dendrites of pyramidal cells (Nolan, et al., Cell, 119:719-732, 2004). The inputs to hippocampal CA1 pyramidal cells are segrated such that the Schaeffer collaterals from CA3 neurons arrive on the distal portion of the dendrite, while perforant pathway connections from entorhinal cortex terminate more proximally. The opening of Ih channels on the distal portion of CA1 dendrites thus functionally disconnects pyramidal cells from CA3 inputs without influencing perforant path connections (Nolan, et al., Cell, 119:719-732, 2004).
As in hippocampus, the HCN1 and HCN2 subunits of the Ih channels are both present in PFC, and likely form heteromers that are highly responsive to cAMP (Chen, et al., J Gen Physiol., 117:491-504, 2001 and Ulens and Tytgat, J Biol. Chem., 276:6069-6072, 2001). Electron microscopic studies have noted a2A-adrenoceptors in post-synaptic spines in monkey PFC (Aoki, et al., Brain Res., 650:181-204, 1994; and Aoki, et al., Cerebral Cortex, 8:269-277, 1998).
HCN1 labeling can similarly be observed in the spines of pyramidal cells in monkey PFC, and indeed, recent EM analyses have shown that HCN1 channels are co-localized with alpha-2A-adrenoceptors in the dendritic spines of PFC pyramidal cells. Electrophysiological and cognitive experiments examined whether a2A-adrenoceptor agonists such as guanfacine may improve PFC cognitive function by reducing cAMP and closing Ih channels, thus strengthening the PFC networks that underlie delay-related cell firing in monkeys performing a spatial working memory task Parallel studies examined whether infusion of the HCN channel blocker, ZD7288, into the rat PFC would improve behavioral measures of working memory performance.
HCN channels and α2A-ARs are co-expressed at exrasynaptic sites (E, F), including the spine neck (G), or where a dendrite tapers outwards, possibly to give rise to a spine (1)). Singly labeled profiles in (ID) attest to the specificity of dual immunolabeling; lead counterstaining was omitted to facilitate detection. Asterisks mark spine apparata. ax, axon; den, dendrite; gl, glial process; sp, spine Scale bars: 200 nm.
The present invention relates to a number of compounds which may be used to modulate Ih channels, preferably including blocking or inhibiting Ih channels to strengthen the connectivity of PFC microcircuits or neurons (prefrontal cortical function) to treat impairment of cognitive function in a patient. Applicants have discovered in animals studies that a compound which functions as an Ih channel blocker may be used to enhance prefrontal cortical function and to be useful to treat cognitive deficiencies, the impairment of cognitive function or to enhance cognitive function in a human, including memory disorders/deficiencies and learning disorders/deficiencies, which may result from aging, trauma, stroke, neurotoxic agents, neurodegenerative disorders or anxiety disorders, including those which are associated with drug-induced states, neurotoxic agents, Alzheimer's disease, and aging. These conditions are readily recognized and diagnosed by those of ordinary skill in the art and treated by administering to the patient an effective amount of one or more compounds according to the present invention. The present invention is also useful for the treatment of memory disorders/deficiencies and/or learning disorders/deficiencies which may be associated with such conditions as attention deficit disorder (ADD), attention deficit disorder with hyperactivity (ADD-HD), autism, and pervasive development disorder (PDD, including PDD-NOS).
In addition, Ih inhibitors or blockers according to the present invention may be used to treat the loss of function in patients from cAMP opening of Ih (HCN) channels and prefrontal cortical impairment (PFC), which are implicated in schizophrenia or certain mood disorders such as depressive disorders. These inhibitors or blockers therefore may be used to treat schizophrenia, and mood disorders, including bipolar disorder, unipolar disorder, dysthymic disorder, post-partum depression, seasonal affective disorder and depression (major depression or major depressive disorder).
Thus, in the present invention, a method of treating cognitive disorders in a patient in thereof comprises administering an effective amount of an inhibitor or blocker of an 1 h channel in said patient. In preferred aspects of the present invention, the Ih channel is HCN1, HCN2 or a heteromer of HCN1 or HCN2. A particularly preferred target of the blockers or inhibitors of the present invention is the Ih channel comprising a heteromer of HCN1 and HCN2.
In certain preferred aspects, the present invention relates to a compound or method using said compound comprising administering an effective amount of a compound according to the structure:
Where R1 is H, or an optionally substituted C1-C3 alkyl, preferably a C2 alkyl (ethyl) group;
R2 is an optionally substituted C1-C3 alkyl group, preferably a methyl group;
R3 is H, an optionally substituted C1-C3 alkyl (preferably methyl), a halogen or O(C1-C3) alkyl;
R4 is an optionally substituted C1-C6 alkyl, C(O)—(C1-C5)alkyl, C(O)-aryl, C(O)O—(C1-C4)alkyl, C(O)O-aryl, or an optionally substituted heterocyclic, aryl or heteroaryl group;
R4a is H or an optionally substituted C1-C6 (preferably a C1-C3) alkyl;
R5, R6 and R7 are each independently H, halogen, an optionally substituted C1-C6 alkyl (preferably, an optionally substituted C1-C3 alkyl), O—(C1-C3) alkyl, optionally substituted heterocyclic, aryl or heteroaryl group;
Y− is an anion of a pharmaceutically acceptable salt (a physiologically acceptable anion, preferably a Cl−, Br−, I−, OAc−);
or a solvate or polymorph thereof, optionally, in combination with a pharmaceutically acceptable carrier, additive or excipient to a patient in need of therapy.
In certain preferred aspects of the present invention, the compound to be used in the present methods is ZD7288, represented by the formula presented hereinbelow.
Additional aspects of the present invention relate to the administration of an Ih channel blocker according to the present invention in combination therapy, for example, with an effective amount of another agent, such as an agent which inhibits cAMP such as guanfacine (N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide) or its pharmaceutically acceptable salt and/or chelerythrine, in its neutral or salt form (which includes all pharmaceutically acceptable salt forms, including the naturally occurring chloride salt form), preferably chelerythrine chloride, in order to potentiate the activity of ZD7288 or one of its analogs or derivatives, thereof.
As used herein, the following terms have the following respective meanings. Other terms that are used to describe the present invention have the same definitions as those generally used by those skilled in the art. Specific examples recited in any definition are not intended to be limiting in any way.
The term “patient” or “subject” refers to a mammal, preferably a human to which one or more of the present methods is applied.
The term “effective” is used in context, to describe an amount of a compound or compound, a component or components or a substance or substances which produce a result intended from the use of that compound(s), component(s) or substance(s).
The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. The compounds of the present invention include all stereoisomers (i.e, cis and trans isomers), tautomers, and all optical isomers of the present compound and related analogs in context (eg., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers, as well as all solvates and polymorphs of the compounds.
The term “Ih channel” refers to a channel formed from one or more hyperpolarization-activated cAMP-regulated cation (HCN) channels. See, Chen, et al., J. Gen. Physiol., 117, 491-503 (May, 2001). Recently, a family of four mammalian genes encoding hyperpolarization-activated cAMP-regulated cation (HCN) channels were cloned. Santoro, et al., Proc. Natl. Acad. Sci. USA, 94, 14815-14820, 1997 and Santoro, et al., Cell, 93, 717-729, 1998. The four genes (H1, H2, H3 and H4) encode highly similar proteins that belong to the voltage-gated K channel superfamily and contain six transmembrane segments, a pore-forming P region and cytosolic NH2 and COOH termini. Jan and Jan, Annu. Rev. Neurosci., 20, 91, (1997). The COOH terminus of the HCN channels also contains a cyclic nucleotide binding domain (CNBD) homologous to those of other cyclic nucleotide binding proteins, including the cyclic nucleotide-gated channels of photoreceptors and olfactory neurons. See Zagota nd Sigelbaum, Annu. Rev. Neurosci., 19, 235-263 (1996).
In aspects of the present invention, compounds according to the present invention block or inhibit L channels, including any one or more of the HCN subunits 1-4, preferably HCN 1 and/or 2 and in particular, the HCN 1 & 2 heteromer (HCN1/HCN2 heteromer Ih channel) as described by Chen, et al., Gem Physiol., 117, 491-503 (May, 2001). HCN 1 and 2 are found in the prefrontal cortex (PFC) and the hippocampus and form a heteromer there. The HCN1/HCN2 heteromer Ih channel is the preferred target for blockers to effect treatment of cognitive disorders according to the present invention.
The term “Ih channel blocker” “Ih channel inhibitor”, “blocker of Ih channels” or “inhibitor of Ih channels” are used interchangeably throughout the present application to describe compounds which directly inhibit or block at least a significant part of the function of Ih channels to generate hyperpolarization-activated cation currents. The term “direct” means that the action of the blocker or inhibitor according to the present invention is directly on the Ih channel, rather than inhibition at another site, which indirectly may cause the release of one or more substances which cause inhibition. By reducing or preventing these cation currents from developing, it is shown that Ih channel blockers may be used to treat cognitive disorders by enhancing cognitive functions such as memory or learning, among others.
“Alkyl” refers to a monovalent hydrocarbon radical, preferably a folly saturated hydrocarbon group, containing carbon and hydrogen which may be a straight chain, branched, or cyclic. Examples of alkyl groups are methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclopentylethyl and cyclohexyl. “Cycloalkyl” groups refer to cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. C1-C6 alkyl groups are preferably used in the present invention. The term “alkyl” also refers to unsaturated alkyls, containing one or more unsaturated groups, within context, and includes alkenyl and alkynyl groups as described hereinbelow, distinguished from aromatic groups, which are fully unsaturated.
The term “substituted” shall mean, within context, a group (substitution group) which is added to a moiety within a compound, e.g. “an optionally substituted alkyl group”. Pursuant to the present invention, within context, the substitution group can be alkyl or alkylene groups containing from 1 to 6 carbon atoms, preferably a lower alkyl containing 1-3 carbon atoms, aryl, substituted aryl, acyl, halogen (i.e., alkyl halos, e.g., CF3), hydroxy, alkoxy, alkoxyalkyl, amino, alkyl and dialkyl amino, acylamino, acyloxy, aryloxy, aryloxyalkyl, carboxyakyl, carboxamido, thio, thioethers, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like
“Substituted alkyl” refers to alkyls (including unsaturated alkyls) as above-described which include one or more substituted groups such an alkyl or alkylene groups containing from 1 to 6 carbon atoms, preferably a lower alkyl containing 1-3 carbon atoms, aryl, substituted aryl, acyl, halogen (i.e., alkyl halos, e.g., CF3), hydroxy, alkoxy, alkoxyalkyl, amino, alkyl and dialkyl amino, acylamino, acyloxy, aryloxy, aryloxyalkyl, carboxyalkyl, carboxamido, thio, thioethers, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. The term “substituted cycloalkyl” has essentially the same definition as and is subsumed under the term “substituted alkyl” for purposes of describing the present invention.
“Aryl” refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Other examples include heterocyclic aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as imidazolyl, furyl, pyrrolyl, pyridyl, thienyl and indolyl, among others. Therefore, “aryl” as used herein includes “heteroaryls” having a mono- or polycyclic ring system which contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, and in which at least one ring of the ring system is aromatic. Heteroatoms are sulfur, nitrogen or oxygen.
“Substituted aryl” refers to an aryl as just described that contains one or more functional groups such as lower alkyl acyl, aryl, halogen, alkylhalos (e.g., CF3), hydroxy, alkoxy, alkoxyalkyl, amino, alkyl and dialkyl amino, acylamino, acyloxy, aryloxy, aryloxyalkyl, carboxyalkyl, carboxamido, thio, thioethers, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like.
“Halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent. The terms “haloalkyl,” “haloalkenyl” or “haloalkynyl” (or “halogenated alkyl”) refers to an alkyl, alkenyl or alkynyl group, respectively, in which at least one of the hydrogen atoms in the group has been replaced with a halogen atom.
“Heterocycle” or “heterocyclic” refers to a carbocylic ring wherein one or more carbon atoms have been replaced with one or more heteroatoms such as nitrogen, oxygen or sulfur. A substitutable nitrogen on an aromatic or non-aromatic heterocyclic ring may be optionally substituted. The heteroatoms N or S may also exist in oxidized form such as NO, SO and SO2. Examples of heterocycles include, but are not limited to, piperidine, pyrrolidine, morpholine, thiomorpholine, piperazine, tetrahydrofuran, tetrahydropyran, 2-pyrrolidinone, δ-velerolactam, δ-velerolactone and 2-ketopiperazine, among numerous others.
“Heteroatom-containing” refers to a molecule or molecular fragment in which one or more carbon atoms is replaced with an atom other carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon. “Substituted heterocycle” refers to a heterocycle as just described that contains one or more functional groups such as lower alkyl, acyl, aryl, cyano, halogen, hydroxy, alkoxy, alkoxyalkyl, amino, alkyl and dialkyl amino, acylamino, acyloxy, aryloxy, aryloxyalkyl, carboxyalkyl, carboxamido, thio, thioethers, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. In other instances where the term “substituted” is used, the substituents which fall under this definition may be readily gleaned from the other definitions of substituents which are presented in the specification as well the circumstances under which such substituents occur in a given chemical compound. One having ordinary skill in the art will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non-aromatic, is determined by the size of the ring, degree of unsaturation, and valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heterocyclic ring is chemically feasible and stable.
“Isostere” refers to compounds that have substantially similar physical properties as a result of having substantially similar electron arrangements.
“Amine” refers to aliphatic amines, aromatic amines (e.g., aniline), saturated heterocyclic amines (e.g., piperidine), and substituted derivatives such as an ably morpoline. “Amine” as used herein includes nitrogen-containing aromatic heterocyclic compounds such as pyridine or purine.
“Aralkyl” refers to an alkyl group with an aryl substituent, and the term “aralkylene” refers to an alkenyl group with an aryl substituent. The term “alkaryl” refers to an aryl group that has an alkyl substituent, and the term “alkarylene” refers to an arylene group with an alkyl substituent. The term “arylene” refers to the diradical derived from aryl (including substituted aryl) as exemplified by 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and the like.
“Alkenyl” refers to a branched or unbranched hydrocarbon group typically although not necessarily containing from 2 to about 12 carbon atoms and at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. Generally, although again not necessarily, alkenyl groups herein contain 2 to about 12 carbon atoms. The term “lower alkenyl” intends an alkenyl group of two to six carbon atoms, preferably two to four carbon atoms.
“Substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom.
“Alkynyl” as used herein is subsumed under the term alkyl and refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 12 carbon atoms and at least one triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl, and the like. Generally, although again not necessarily, alkynyl groups herein contain 2 to about 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of two to six carbon atoms, preferably three or four carbon atoms. “Substituted alkynyl” refers to alkynyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkynyl” and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom.
“Alkoxy” as used herein refers to an alkyl group bound through an ether linkage; that is, an “alkoxy” group may be represented as —O-alkyl where alkyl is as defined above. A “lower alkoxy” group intends an alkoxy group containing one to six, more preferably one to four, carbon atoms.
The term “coadministration”, “coadministered” or “combination therapy” is used to describe a therapy in which at least two active compounds in effective amounts are used to enhance cognitive function or to treat a cognitive deficit using an Ih blocker or inhibitor in combination with guanfacine or its pharmaceutically acceptable salt or chelerythrine (as its neutral or salt form) as otherwise described herein at the same time. Although the term coadministration preferably includes the administration of two active compounds to the patient at the same tire, it is not necessary that the compounds be administered to the patient at the same time, although effective amounts of the individual compounds will be present in the patient at the same time.
The term “cognitive function” is used to describe an endeavor or process by a patient or subject that involves thought or knowing. The diverse functions of the association cortices of the parietal, temporal and frontal lobes, which account for approximately 75% of all human brain tissue, are responsible for much of the information processing that goes on between sensory input and motor output. The diverse functions of the association cortices are often referred to as cognition, which literally means the process by which we come to know the world. Selectively attending to a particular stimulus, recognizing and identifying these relevant stimulus features and planning and experiencing the response are some of the processes or abilities mediated by the human brain which are related to cognition. Compounds and compositions of the present invention may be used to enhance cognition or reduce impairment of cognitive function.
Impairment of cognitive function (“cognitive disorder”) includes memory disorders and learning disorders, which are treatable according to the present, including those disorders that result from aging, trauma, stroke, neurodegenerative disorders or anxiety disorders. Examples of neurodegenerative disorders include, but are not limited to, those associated with drug-induced states, neurotoxic agents, Alzheimer's disease, and aging. These conditions are readily recognized and diagnosed by those of ordinary skill in the art and treated by administering to the patient an effective amount of one or more compounds according to the present invention. The present invention is also useful for the treatment of attention deficit disorder (ADD), attention deficit disorder with hyperactivity (ADD-HD), autism, pervasive development disorder (PDD, including PDD-NOS), learning disabilities and disorders associated with PFC dysfunction such as and schizophrenia and bipolar disorder. etc.
“Anxiety disorders” include affective disorders such as all types of depression, bipolar disorder, cyclothymia, and dysthymia, anxiety disorders such as generalized anxiety disorder, panic, phobias and obsessive-compulsive disorder, stress disorders including post-traumatic stress disorder, stress-induced psychotic episodes, psychosocial dwarfism, stress headaches, and stress-related sleep disorders, and can include drug addiction or drug dependence.
An “anion of a pharmaceutically acceptable salt” refers to a negatively charged species otherwise represented as Y− in the structures according to the present invention. The anions of pharmaceutically acceptable salts, include, for example, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3 naphthoate)] anions, among numerous others.
The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled-release formulations. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously.
Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may also be administered topically. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
The amount of a compound used in a pharmaceutical composition of the instant invention that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration and may vary widely. Preferably, the compositions should be formulated to contain between about 10 milligrams to about 500 milligrams of active ingredient
It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.
Synthesis of Compounds According to the President Invention
The compounds according to the present invention may be synthesized by synthetic methods which are well-known in the art. The synthesis of compounds according to the present invention may proceed by following the general procedure for such compounds as presented in U.S. Pat. No. 5,223,505, relevant portions of which are incorporated by reference herein, PCT application WO 90/12790, a counterpart application to U.S. Pat. No. 5,223,505 and published Japanese patent application 2004-59465. All of the methods for synthesizing compounds according to the present invention are well-known in the art or are adapted from well-known methods in the art without undue experimentation.
In addition to the patent publication references cited above, compounds according to the present invention may be synthesized by condensing a substituted aniline compound onto a pyrimidine intermediate containing a leaving group (halogen such as Br or I) at the 6 position of a substituted 4-aminopyrimidine ring or related pyrimidine analog. The starting materials for such a synthesis may be purchased commercially or produced using methods well known in the art. Alkylation of the appropriate amino group of the resulting intermediate is effected using one or more standard alkylating agents. The resulting compound may be used directly or the counterion may be changed (ion-exchange) to reflect desired solubility/administration parameters.
The invention is described further in the following examples, which are illustrative and in way limiting.
The present invention illustrates the effects of Ih channel blockade in prefrontal cortex on 1) cognitive performance in rats engaged in a spatial working memory task, and 2) firing patterns of prefrontal cortical neurons in monkeys performing a spatial working memory task.
Rats (n=5) were tested on the spatial delayed alternation task in a T maze, a classical test of prefrontal cortical function in rodents (Larsen and Divac, Physiolog Psychol, 6:15-17, 1978). Rats were trained on the task, and then implanted with cannula aimed at the prelimbic prefrontal cortex as described in (Ramos, et al., Neuron, 40:835-845, 2003). Following recovery from surgery, rats were infused with saline (0.5 μl), the Ih channel blocker ZD7288 (0.0001 μg/0.5 μl), the cAMP analog Sp-cAMPS (0.21 nmol/0.5 μl), or a combination of ZD7288 (0.0001 μg)+Sp-cAMPS (0.21 nmol). There was at least one week washout between drug treatments, and a within subjects comparison was performed (2 way analysis of variance with repeated measures with user defined contrasts). As can be seen in
Similar enhancing effects have been observed previously with infusions of the α2A agonist guanfacine into prefrontal cortex of monkeys (Mao et al, Biol Psychiatry 46:1259-1265, 1999) and rats (e.g.
Spatial working memory is maintained by spatially tuned, recurrent excitation within networks of prefrontal cortical (PFC) neurons, evident during delay periods in working memory tasks. Stimulation of post-synaptic α2A adrenoceptors (α2A-ARs) is critical for working memory. The following examples show that α2A-AR stimulation strengthens working memory through inhibition of cAMP, closing Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels and strengthening the functional connectivity of PFC networks. Ultrastructurally, HCN channels and α2A-ARs were colocalized in dendritic spines in monkey PFC. In electrophysiological studies, either α2A-AR stimulation, cAMP inhibition or HCN channel blockade enhanced spatially tuned delay-related firing of PFC neurons in monkeys performing a working memory task. Conversely, delay-related network firing collapsed under conditions of excessive cAMP. In behavioral studies, HCN channel blockade or knockdown of HCN1 channels in rat PFC improved working memory performance. These data reveal a powerful mechanism for rapidly altering the strength of working memory networks in PFC.
Single Neuron Recording and Iontophoresis in Monkeys Performing a Spatial WM task
Studies were performed on 4 adult male rhesus monkeys trained on the spatial ODR task (
In Vitro Recording from PFC Slices
Methods for extracellular multiple unit and intracellular recording in ferret PFC slices have been detailed elsewhere (Shu et al., 2003). Slices (0.4 mm) from 2-4 month old ferret PFC were maintained in either an interface or submerged chamber (35-36° C.) in a slice solution containing (in mM): NaCl, 126; KCl, 3.1; MgSO4, 1; NaHPO4, 1.25; CaCl2, 1; NaHCO3, 26; dextrose, 10, and aerated with 95% O2, 5% CO2 to a final pH of 7.4. Simultaneous extracellular multiple unit and intracellular recordings were performed in layer 5 with the electrodes approximately 100 mm of one another. Intracellular micropipettes contained 2 M potassium acetate, while whole cell recording pipettes contained KGluconate 140, KCl 3, MgCl2 2, Na2ATP 2, HEPES 10, and EGTA 0.2.
Assessment of Spatial WM Performance in Rats
Male Sprague Dawley rats (240-260 g; Taconic, Germantown, N.Y.) were trained on the delayed alternation task as described in Ramos et al., 2003. Guide cannula were implanted dorsal in prelimbic PFC (AP: +3.2 mm; ML: ±0.75 mm; DV: −4.2 mm). For infusions, needles reached to 4.5 mm DV; infused at 0.25 μL/min for 2 min. Drug or vehicle was administered in a counterbalanced order with at least 1 week between infusions. ZD7288 was dissolved in saline to a dose of 0.0001 μg/0.5 μL. Sp-cAMPS was dissolved in sterile phosphate-buffered saline at 0.21 nmol, a dose with no effect on its own, but sufficient to reverse the effects of guanfacine (Ramos et al., 2006). The experimenter was blind to treatment.
Viral Knockdown of HCN1
The coding region of HCN1 was amplified from rat brain cDNA, cloned into pSTBlue-1 (Novagen, San Diego Calif.), sequenced, and subcloned into pAAV-MCS (Stratagene, La Jolla, Calif.) to create pAAV-HCN1. A vector containing an H1 promoter for shRNA expression and a CMV promoter for lacZ expression flanked by AAV ITR sequences (pAAV-IacZ-shRNA) was created to allow viral expression of shRNAs. Two sets of shRNAs constructs (shRNA-HCN1.1 and shRNA-HCN1.2) directed against HCN1 were generated and tested in HEK293 cells by co-transfection with pAAV-HCN1. A control shRNA viral construct containing a scrambled sequence (shRNA-scrHCN1) was created with an identical nucleotide composition as the shRNA-HCN1.1 target sequence with no homology to any mammalian gene in the Genbank database. AAV2 virus was produced by transfection of HEK293 cells with pAAV-HCN1 or one of the pAAV-IacZshRNA constructs, and pDG essentially as described by (Auricchio et al., 2001). Viral titers were determined by determining viral genome copy number and by infectious titer assays in HEK293 cells.
Rats were implanted with cannula, and trained until achieving baseline performance of ˜70% correct for at least 5 consecutive days. The active or scrambled virus (5 μL) was infused into the prelimbic PFC at a rate of 0.25 μL/min WM was assessed the following day and for the subsequent 3 weeks. Rats were perfused and viral transfection was confirmed in PFC with a monoclonal antibody against B-galactosidase (Mouse anti B-gal 1:400, overnight, RT; Promega Corp., Madison, Wis.) followed by a secondary antibody (Goat anti mouse conjugated to Alexa Flour® 488, 1:400, 4 h, RT; Invitrogen/Molecular Probes, Carlsbad, Calif.). Antibodies were prepared in 0.1M PB containing 0.1% Triton X and 2% normal goat serum. All behavioral studies were performed blind to treatment.
HCN and α2A-AR Localization in Monkey PFC-Electron Microscopic Studies
Monkey PFC tissue (n=3) was prepared as described in the Supplemental Material. For single HCN1, HCN-pore or α2A-AR immunocytochemistry, primary antibodies were complexed with nanogold conjugates, either directly or using biotinylated bridging antibodies. F dual immunolabeling, we used combinations of enzymatic and/or gold-based immunotechniques and reversal of the immunocytochemical sequence (
Monkey Physiology: PFC Neurons Exhibit Enhanced Spatial Tuning when cAMP-HCN Signaling is Inhibited by α-2A-ARs
Monkeys performed an oculomotor spatial delayed response (ODR) task, illustrated in
Effects of α2A-AR Stimulation on PFC Neurons with WM Activity
Intra-PFC administration of the α2A-AR agonist, guanfacine, improves WM performance (Mao et al., 1999). However, guanfacine has never been examined for its effects on PFC neuronal firing. In the present study, guanfacine was applied iontophoretically to 35 neurons with spatial delay-related activity. Low doses of guanfacine (5-15 nA) significantly enhanced delay-related activity for the preferred direction in 28 out of 35 cases, while having no effect on neuronal activity of the remaining 7 neurons. In contrast, high doses of guanfacine (20-50 nA) suppressed delay-related activity in 9 out of 9 neurons, perhaps due to stimulation of pre-synaptic α2A-ARs, reducing endogenous NE release.
Previous studies demonstrated that iontophoretic application of yohimbine, an α2-AR antagonist, suppressed delay-related firing in PFC neurons (Li et al., 1999). Replicating these previous reports, iontophoresis of yohimbine (15 nA) suppressed delay-related activity for the preferred direction, thus eroding spatial mnemonic tuning (P<0.0001,
Influence of cAMP on PFC Neurons Engaged in a WM Task
Behavioral studies in rats have shown that amplification of cAMP actions with Sp-cAMPS, a cAMP analog, impaired spatial WM, while inhibition of cAMP actions with Rp-cAMPS ameliorated WM deficits. Consistent with these behavioral results, we found that iontophoresis of Sp-cAMPS (10 nA) significantly decreased delay-related firing for the preferred direction (P<001,
Etazolate is a PDE4 inhibitor that increases cAMP levels by inhibiting the breakdown of endogenously produced cAMP. Iontophoretic application of etazolate (25 nA) had highly consistent suppressing effects on spatial mnemonic activity in 10 of 12 PFC neurons. One example is shown in
In contrast to Sp-AMPS and etazolate, iontophoresis of the cAMP inhibitor, Rp-cAMPS (40-50 nA) specifically increased delay-related firing for the preferred direction in 8 of 12 neurons (P<0-001,
Guanfacine Acts Via Inhibition of cAMP
The enhancing effects of guanfacine on WM in rats has been reversed by co-infusion of Sp-cAMPS (Ramos et al., 2006). We examined whether the enhancing effects of guanfacine on PFC neuronal firing are similarly reversed by Sp-cAMPS.
As observed above, iontophoresis of guanfacine (10 nA) significantly increased delay-related firing for the preferred direction (P<0.001,
A putative downstream target of cAMP is the HCN channel. cAMP opens HCN channels, inducing an Ih current which in turn reduces membrane resistance. We examined the role of Ih in WM by iontophoresing the HCN channel blocker, ZD7288.
Low doses of ZD7288 (10 nA), like guanfacine or Rp-cAMPS, significantly increased delay-related firing for the preferred direction in 19 of 27 neurons. As with guanfacine, ZD7288 effects were most obvious in neurons with weak spatial tuning, however, small but consistent effects were also observed in well-tuned neurons with robust firing for the preferred direction under control conditions. For example, in
A functional link between HCN channels and α2-ARs was examined by observing whether ZD7288 could reverse the effects of α2-AR blockade (
We also examined the functional interactions between HCN channels and cAMP. As shown in
The cellular and network consequences of modulation of HCN channels was examined with intracellular and whole cell recordings from layer 5 pyramidal cells simultaneously with extracellular multiple unit recordings in the ferret PFC in vitro (n=55, see
The next study tested the hypothesis that reducing Ih in rat prelimbic PFC (
The first experiment tested whether infusion of ZD7288 into prelimbic PFC would improve performance as previously seen with guanfacine (Ramos et al., 2006) and Rp-cAMPS (Ramos et al., 2003). Pilot experiments explored a wide range of ZD7288 doses (0.00001-0.1 μg/0.5 μl). Improvement was only observed in the low dose range; thus, the 0.00011 g dose became the focus of the present study. ZD7288 was challenged with a low dose of the cAMP analog, Sp-cAMPS (0.21 nmol//0.5 μL) chosen to have no effect on its own. Results are shown in
In the second experiment, HCN1 expression was knocked down by RNA interference in prelimbic PFC through infusion of one of two short hairpin expressing viral constructs, shRNA-HCN1.1 or shRNA-HCN1 0.2 (
Ultrastructurally, HCN1 and the pore region of HCN (corresponding to HCN1 and/or HCN2 subunits in the cortex; (Notomi and Shigemoto, 2004) were predominantly detected in dendritic spines and the shafts of pyramidal dendrites (
α2A-AR-immunoreactive profiles in the neuropil included the spine-laden pyramidal dendrites, besides axonal and glial localization. Distal dendrites showed plasmalemmal but also cytoplasmic labeling, indicative of a rapid turnover from the plasma membrane. Similar to HCN, α2A-AR-immunoparticles in spines marked extrasynaptic and perisynaptic membranes flanking asymmetric synapses. In addition, we observed α2A-AR labeling intracellularly and rarely within the postsynaptic specialization per se. It is worth noting that both the head and the neck portion of spines were immunoreactive for α2A-ARs, as with HCN channels (
Dual immunolabeling confirmed the co-expression of HCN1 channels and α2A-ARs in spines and dendritic shafts at sites where emerging spines would come into focus. HCN1/α2A-AR labeling involved both the spine head and the neck portion. This pattern was reproduced with reversal of the immunocytochemical sequence, and with both peroxidase/gold (
The current data provide the very first evidence that HCN channels have powerful effects on PFC network firing properties and cognitive performance in animals performing WM tasks. Blockade of HCN channels with ZD7288 promoted persistent network activity and enhanced the spatial mnemonic firing of PFC neurons. Similar enhancing effects were observed with stimulation of α2A-ARs or inhibition of cAMP. Conversely, increasing cAMP signaling—either directly with Sp-cAMPS, or indirectly via blockade of α2-ARs or PDE4 inhibition—dramatically suppressed delay-related firing. Blockade of HCN channels restored mnemonic activity in cells with excessive cAMP signaling induced by either blockade of α2-ARs or PDE4 inhibition, thus demonstrating a functional interaction between α2-ARs, endogenous cAMP, and HCN modulation of PFC neuronal firing. Similar effects were observed at the behavioral level, where HCN channel blockade or HCN1 channel knockdown in PFC improved spatial WM performance. Ultrastructural localization of HCN channels and α2A-ARs indicates that they are ideally situated to modulate synaptic inputs onto PFC pyramidal neurons.
Imunoelectron microscopy revealed HCN channel expression in spines of pyramidal dendrites in layers I-m of the primate PPC. Gold particles marked both extrasynaptic and perisynaptic membranes flanking asymmetric, presumed excitatory synapses. More interestingly, the spine neck was often HCN-immunoreactive. These data indicate that HCN channels are ideally suited for gating glutamatergic transmission mediated by axospinous synapses, including cortico-cortical inputs of the superficial PFC. When opened in the presence of cAMP, HCN channels would lower membrane resistance and shunt inputs to the spine. Due to the small cytosolic volume, HCN channels in the spine head—and especially those in the spine neck—are capable of “sensing” minute neurochemical changes in the milieu caused by the downstream effects of “co-localized” neurotransmitter receptors. Our ultrastructural data suggest that one such candidate receptor in PFC is the α2A-AR. It is noteworthy that the latter was found not only in the head but also in the neck of spines on both perisynaptic and extrasynaptic membranes, and dual labeling confirmed the spatial co-expression of HCN1 and α2A-ARs. α2A-ARs inhibit cAMP production via Gi signaling and thus could have a powerfuil influence on the open-state of nearby HCN channels. Inadequate stimulation of α2A-ARs would result in elevated cytosolic cAMP levels, opening Ih channels, and selectively disconnecting axospinous inputs to cortical pyramids. Therefore, cytosol compartmentalization in the spine and the co-expression of HCN channels with α2A-ARs on spine membranes may provide a cellular basis for altering the strength of excitatory transmission at individual axospinous synapses, thus modulating circuit connectivity in PFC.
α2A-AR Inhibition of cAMP-HCN Signaling Enhances the Spatially Tuned, Delay-Related Firing of PFC Pyramidal Cells
The functional implications suggested by immunoelectron microscopy were supported by our physiological data. Delay-related firing arises from reverberating, excitatory circuits in PFC, and depends on the functional connectivity of neurons with shared spatial tuning. The current study found that agents, which 1) stimulate α2A-ARs, 2) inhibit cAMP signaling or 3) block HCN channels, all increase delay-related firing for the preferred direction, consistent with increased functional connectivity under conditions where HCN channels are closed. Conversely, delay-related firing collapsed in the presence of agents that blocked α2A-ARs or increased cAMP signaling, consistent with reduced functional connectivity when cAMP opens HCN channels. These modulatory effects were very powerful: extremely low doses selectively altered delay-related firing for the preferred direction, while slightly higher doses had more generalized effects on firing, consistent with perturbations in network firing. Parallel results were observed at the behavioral level, where PFC infusions of guanfacine (Ramos et al., 2006) or ZD7288, or knockdown of HCN1 expression in PFC, all improved WM performance, while agents that blocked α2A-ARs (Li and Mei, 1994) or accentuated cAMP signaling (Taylor et al., 1999) impaired WM. It is remarkable to have such confluence between behavioral and electrophysiological findings.
ZD7288 is currently the most selective HCN antagonist available, and as such, has become the standard pharmacological method for assessing Ih mechanisms, e.g. (Fan et al., 2005). However, it has recently been noted that at higher doses ZD7288 becomes nonselective, producing ALA and NMDA glutamate receptor blockade (Chen, 2004). The inhibition of cell activity at higher ZD7288 concentrations (25-40 nA) in the current study could be consistent with reduced glutamate receptor excitation. A similar profile was observed in our behavioral data, where pilot studies showed that higher doses of ZD7288 were ineffective, whereas a very low dose consistently improved WM performance.
Physiological interactions between α2A-ARs and HCN channels were observed, consistent with their co-expression in spines: The collapse in memory fields induced by the α2A-AR antagonist, yohimbine, was reversed by the HCN channel blocker, ZD7288. HCN channel blockade similarly reversed the suppressive effects of the PDE4 inhibitor, etazolate. Thus, iontophoresis of yohimbine, Sp-cAMPS or etazolate, all induced an immediate collapse in memory fields, likely due to a reduction in recurrent excitatory drive in the PFC network. The blockade of HCN channels with ZD7288 restored a normal firing pattern. It should be noted that these agents are not likely competing at the same individual channel; rather, it is likely that ZD7288 and Sp-cAMPS (or cAMP per se) each alter the open state of a subset of HCN channels, and we record the integration of this population response. It is striking that very low doses can have such rapid and robust effects on cell firing, revealing a powerful mechanism for the dynamic regulation of PFC microcircuits.
Recent studies of HCN1 knockout mice have revealed distinct roles of Ih in cerebellum and hippocampus, and it is instructive to compare the current results with these important findings. In cerebellar Purkinje cells, HCN1 channels mediate an inward current that stabilizes the integrative properties of the cell and ensures that their input-output function is independent of previous activity (Nolan et al., 2003). HCN1 currents allow Purkinje cells to integrate information very quickly, consistent with the rapid time scale of cerebellar mechanisms (ibid). However, the influence of HCN channels in cerebellum occurs only when Purkinje cells are hyperpolarized. In contrast, pyramidal cells in hippocampus and PFC have a lower resting membrane potential, and thus HCN channels may play a role under resting conditions in these cells (Nolan et al., 2004). Pyramidal cells in PFC and hippocampus share other properties: HCN1 channels are localized on their distal dendrites, and, when opened, appear to shunt synaptic inputs onto those distal locations (Magee, 1999; Nolan et al., 2004). However, there are important differences between CA1 and PFC neurons as well. In hippocampus, high levels of cAMP are essential for long-term potentiation and memory consolidation, leading to long-lasting changes in synaptic architecture (cAMP also plays a beneficial role when hippocampus interacts with PFC under conditions when delay lengths are very long, see (Runyan and Dash, 2005). In contrast, the WM operations of the PFC depend on the transient activation of microcircuits that are disrupted by high levels of cAMP. Thus, cAMP/HCN signaling may play an especially important role in PFC.
The examples further indicate that Gs-coupled receptors may temporarily suppress neurotransmission by activating cAMP production, opening HCN channels and shunting synaptic inputs. For example, dopamine D1 receptors (D1Rs) are coupled to Gs and are also concentrated on spines in superficial PFC (Smiley et al., 1994). We have recently observed that moderate levels of D1R stimulation suppress firing for nonpreferred spatial directions via a cAMP-mediated mechanism (Vijayraghavan et al., 2007). It is not known if this suppression involves opening of HCN channels. If so, it is possible that α2A-ARs amplify inputs for preferred directions by selectively closing HCN channels on spines receiving inputs from neurons with shared spatial preferences, while D1Rs may shunt inputs from neurons tuned to nonpreferred directions. Thus, the open state of HCN channels may determine which pattern of microcircuits are functionally connected at any one time to appropriately regulate behavior and thought based on immediate cognitive demands.
αa2A/cAMP/HCN signaling in superficial cortical layers likely regulates the strength of PFC function based on the animal's state of arousal. Low levels of NE cell firing during drowsy conditions (Aston-Jones et al., 1999) may lead to insufficient NE stimulation of α2A-ARs, inadequate inhibition of cAMP, and impaired WM. Conversely, exposure to uncontrollable stress impairs WM via excessive catecholamine release (Arnsten, 2000). Similar effects are observed at the cellular level, where memory fields collapse under neurochemical conditions induced by stress: e.g. etazolate, Sp-cAMPS, (
The previously described examples demonstrate that HCN1 or HCN1/HCN2 heteromers in spines of pyramidal dendrites in the superficial layers of primate PFC are spatially co-expressed with the α2A-AR, thus providing a potent substratum for functional interaction. Electrophysiological and cognitive experiments support a model where α2A-AR agonists such as guanfacine improve PFC cognitive function by inhibiting the production of cAMP, closing HCN channels, and strengthening the PFC networks that underlie delay-related cell firing in monkeys performing a spatial WM task.
These data indicate that the enhancing effects of guanfacine result from closure of Ih channels via inhibition of cAMP production. Given that guanfacine is currently in use for the treatment of neuropsychiatric and neurologic disorders involving weakened prefrontal cortical function—Attention Deficit Hyperactivity Disorder and Tourettes Syndrome (Scahill et al, Amer J Psychiatry, 158:1067-1074, 2001), mild traumatic brain injury (McAllister et al, Brain Inj, 18:331-350 2004), schizophrenia-related illness (Friedman et al, Guanfacine treatment of cognitive impairment in schizophrenia A pilot study. Neuropsychopharmacology, 2001), post-traumatic stress disorder (Horrigan, J Amer Acad Child Adol Psychiatry, 35:975-976, 1996)—these data indicate that agents according to the present invention that block Ih channels in the prefrontal cortex would have therapeutic effects.
It is to be understood by those skilled in the art that the foregoing description and examples are illustrative of practicing the present invention, but are in no way limiting. Variations of the detail presented herein may be made without departing from the spirit and scope of the present invention as defined by the following claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/02527 | 1/31/2007 | WO | 00 | 12/22/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60763802 | Jan 2006 | US |
