Patent 6,884,596

1. Field of the Invention

The present invention relates generally to the field of medicine and, more specifically, to therapeutic compositions and methods relating to Prolactin Releasing Peptide (PrRP).

2. Background Information

Sleep is a naturally occurring, periodic, reversible state of unconsciousness that is ubiquitous in mammals and birds, although its precise function is not known. The importance of sleep is suggested by its homeostatic regulation: the longer an animal is awake, the more it needs to sleep.

In humans, obtaining less than the required number of hours of sleep, particularly over several nights, leads to a decreased ability to retain new information, impaired productivity, altered mood, lowered resistance to infection and an increased susceptibility to accidents. Sleep-related traffic accidents annually claim thousands of lives, and operator fatigue has also been shown to play a contributory role in airplane crashes and other catastrophic accidents.

Besides lifestyle factors, a variety of physiological and psychological disorders can affect sleep patterns. The most common sleep disorder is primary insomnia, or a difficulty in initiating or maintaining sleep, which affects a large percentage of the population at some point in their lives. Other common sleep disorders include hypersomnia, or excessive daytime sleepiness, and narcolepsy, which is characterized by sudden and irresistible bouts of sleep.

Currently available drugs used to modulate wakefulness and sleep, such as drugs that induce sleep, prolong wakefulness, or enhance alertness, suffer from a number of shortcomings. For example, available sleep-inducing drugs often do not achieve the fully restorative effects of normal sleep. Often such drugs cause undesirable effects upon waking, such as anxiety or continued sedation. Furthermore, many of the currently available drugs that modulate sleep and wakefulness are addictive or have adverse effects on learning and memory.

Thus, there exists a need to identify new therapeutic agents that can be used to promote wakefulness and sleep. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides methods of screening for a compound for promoting wakefulness in a mammal. The method is practiced by providing a compound that is a PrRP receptor agonist and determining the ability of the compound to promote wakefulness.

In one embodiment, the method is practiced by providing a compound that promotes a predetermined signal. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP promotes a predetermined signal, and identifying a compound that promotes the predetermined signal.

In another embodiment, the method is practiced by providing a compound that binds to PrRP receptor. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP binds to the PrRP receptor and identifying a compound that binds to PrRP receptor.

In a further embodiment, the method is practiced by providing a compound that promotes interaction of PrRP receptor with an AMPA receptor associated protein. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP promotes interaction of PrRP receptor with an AMPA receptor associated protein and identifying a compound that promotes this interaction.

The invention also provides a method of promoting wakefulness in a mammal. The method is practiced by administering to a mammal an effective amount of a PrRP receptor agonist.

The invention further provides methods of screening for a compound for promoting sleep in a mammal. The methods are practiced by providing a compound that is a PrRP receptor antagonist and determining the ability of the compound to promote sleep.

In one embodiment, the method is practiced by providing a compound that reduces a predetermined signal. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein a PrRP promotes a predetermined signal and identifying a compound that reduces the predetermined signal.

In another embodiment, the method is practiced by providing a compound that reduces binding of a PrRP receptor agonist to PrRP receptor. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP binds to said PrRP receptor and identifying a compound that reduces binding of the PrRP receptor agonist to PrRP receptor.

In a further embodiment, the method is practiced by providing a compound that promotes interaction of PrRP receptor with an AMPA receptor associated protein. The compound is identified by contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP promotes interaction of PrRP receptor with an AMPA receptor associated protein and identifying a compound that reduces the interaction.

The invention further provides a method of promoting sleep in a mammal. The method is practiced by administering to a mammal an effective amount of a PrRP receptor antagonist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the determination that PrRP receptor modulation alters the activity of the reticular thalamic nucleus (RTN), a region of the brain implicated in sleep rhythms, attention processing and absence seizures, through a functional interaction between the PrRP receptor (GPR10), and Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The inventors have determined that PrRP receptor activation in response to PrRP receptor agonists specifically reduces AMPA receptor mediated oscillatory activity in the RTN, effectively suppresses absence seizures in mammals. The inventors have further determined that agonist binding to PrRP receptor effectively promotes wakefulness in mammals.

Thus, based on the determination of an important pharmacological role of PrRP, and its underlying molecular mechanism, the invention provides compounds and related therapeutic methods for suppressing absence seizures and promoting wakefulness and sleep in mammals. The compounds and therapeutic methods of the invention can thus be used in the therapy of epilepsies and other diseases associated with absence seizures, and in promoting wakefulness and sleep in normal individuals and those having sleep disorders, such as insomnia and narcolepsy. Additionally, the invention provides methods of rapidly screening for compounds that modulate AMPA receptor signaling and compounds that control absence seizures and promote wakefulness and sleep. The compounds so identified will be useful in the control of absence seizures and promoting wakefulness and sleep, as well as in the prevention and treatment of conditions associated with tissues in which GPR10 is expressed, or in which known anti-epileptic drugs are effective.

In one aspect, the invention provides a method of controlling absence seizures. The method involves administering to a mammal susceptible to absence seizures an effective amount of a PrRP receptor agonist, such as PrRP or a functional analog thereof. As used herein, the term "mammal susceptible to absence seizures," refers to a human, veterinary animal or laboratory animal (e.g. non-human primate, rodent, feline or canine) that exhibits, can be induced to exhibit, or is at high risk of developing, absence seizures.

Absence seizures are brief attacks of impaired consciousness that can be distinguished from other forms of seizures both by their distinct electroencephalographic (EEG) patterns and by their response to pharmacological agents. By EEG, absence seizures are associated with bilateral synchronous and regular spike and wave discharges (SWD) with a frequency of 2.5 to 4 c/s, which start and end abruptly. Pharmacologically, absence seizures generally respond to the drugs ethosuximade, valproate and trimethadione, but are worsened by anti-convulsants such as carbamazepine and phenytoine which are effective in treating convulsive seizures.

In humans, several clinically recognized epilepsy syndromes, including childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, myoclonic absence epilepsy, and eyelid myoclonia with absences, are associated with absence seizures. Epileptic syndromes are commonly classified according to the International Classification of Epileptic Syndromes proposed by the International League against Epilepsy in 1989.

Epidemiological studies have identified several predictive factors for the development of absence seizures, including past history of either febrile convulsions or generalized tonic-clonic seizures (GTCS), and family history of epilepsy or febrile convulsions (see, for example, Covanis et al., Seizure 1:281-289 (1992)). Because of the clear genetic predisposition for absence epilepsies, those skilled in the art understand that it will also be possible to determine susceptibility to absence seizures by genetic or biochemical profile.

Accordingly, a human "susceptible to absence seizures," can be a human exhibiting absence seizures, such as a human diagnosed with an epilepsy syndrome characterized by absence seizures, or a human considered to be at high risk of developing absence seizures.

A variety of non-human mammals that exhibit, or can be induced to exhibit, behavioral, electrographic and pharmacological characteristics of absence seizures in humans are known in the art. Such mammals are also considered herein to be "mammals susceptible to absence seizures." For example, Genetic Absence Epilepsy Rats from Strasbourg, or GAERS, exhibit behavioral and EEG patterns during spike and wave discharges (SWD) that are similar to those observed in humans during absence seizures (see Danober et al., Prog. Neurobiol. 55:27-57 (1998)). Other genetic models of absence epilepsy include the lethargic (lh/lh) mutant mouse (see Hosford et al., Epilepsia 38:408-414 (1997)), the WAG/Rij strain of rats (see Coenan et al., Epilepsy Res. 12:75-86 (1992)), and the tremor (tm/tm) mutant rat (Hanaya et al., Epilepsia 36:938-942 (1995)), which have been successfully used to predict or confirm the effects of a variety of anti-epileptic drugs in controlling absence seizures in humans.

Other relevant mammalian models of absence seizures in humans include pharmacological models, in which absence seizures are induced in laboratory animals, such as rodents, cats and primates, by administration of pentylenetetrazol, penicillin, gamma-hydroxybutyrate or GABA agonists (for a review, see Snead, Epilepsia 29:361-368 (1988)). Additionally, absence seizures can be induced in primates by thalamic stimulation (see, for example, David et al., J. Pharmacol. Methods 7:219-229 (1982)).

As used herein, the term "controlling," in relation to absence seizures, refers to a reduction in the frequency, duration, number or intensity of absence seizures in a treated mammal, as compared with the frequency, duration, number or intensity of absence seizures expected or observed without treatment. A determination of whether absence seizures are "controlled" by treatment can be made, for example, by direct observation, by self-reporting, or by examining on an EEG readout the frequency, duration, number or intensity or duration of spike and wave discharges (SWD).

An amount of a pharmaceutical composition effective to control absence seizures is an amount effective to reduce the determined parameter (e.g. frequency, duration, number or intensity of absence seizures or SWD) by at least 10%. Preferably, the determined parameter will be reduced by at least 20%, more preferably at least 50%, such as at least 80%, in at least some treated mammals. Accordingly, a treatment that controls absence seizures will be useful in improving the quality of life in the treated mammals. Further description of effective amounts, formulations and routes of administration of the pharmaceutical compositions useful in the methods of the invention is provided below.

In another aspect, the invention provides a method of promoting wakefulness. The method involves administering to a mammal an effective amount of a PrRP receptor agonist. A mammal to be treated with a PrRP receptor agonist is generally in need of wakefulness and would benefit physically or psychologically from increased wakefulness. For example, increased wakefulness may be desired in an individual having sleepiness, a tendency to fall asleep, or having a sense of excessively deep sleep. A need for wakefulness can arise in an individual having or absent of a sleep-related disorder. For example, an individual may desire wakefulness to enhance performance in mental or physical activities, such as long distance driving, shift work and study. A need for wakefulness can also be caused by a disorder of excessive daytime sleepiness. Sleep apnea, narcoplepsy, idiopathic hypersomnia and psychogenic hypersomnia are examples of common disorders which lead to daytime sleepiness. Other causes of daytime sleepiness include, for example, sleep apnea, obesity, sleep deprivation, and adverse drug reactions.

Idiopathic hypersomnia is a disorder of excessive diurnal and nocturnal sleep characterized by virtually constant sleepiness, lengthy but nonrefreshing naps, prolonged night sleep, major difficulty with morning awakening, and sometimes sleep drunkeness. Idiopathic hypersomnia appears to have familial incidence and is associated with the presence of the HLA antigen HLA-DR5. Therefore, those skilled in the art will understand that it will be possible to determine susceptibility to hypersomnia by genetic or biochemical profile.

Narcolepsy is characterized by excessive daytime sleepiness, often with involuntary symptoms of reduced wakefulness including, for example, cataplexy, which is muscle weakness or paralysis in response to sudden emotion, sleep paralysis, which is the inability to move or call out when first awake, and hallucinations. Pharmacologically, treatment of narcolepsy involves separate treatments for sleep attacks and cataplexy. Epidemiological studies have identified several predictive factors for the development of narcolepsy, including a history of cataplexy. A genetic predisposition to narcolepsy can be indicated by the presence of HLA allele DQB1*0602. Therefore, those skilled in the art understand that it will be possible to determine susceptibility to narcolepsy by genetic or biochemical profile.

A variety of non-human mammals that exhibit, or can be induced to exhibit, behavioral, electrographic and pharmacological characteristics of narcolepsy in humans are known in the art. For example, canine narcolepsy is a naturally-occurring animal model of the human disorder (Riehl, et al., Neuropsychopharmacology, 23:34-35 (2000)). Canine models exhibit short sleep latency, fragmented sleep patterns, cataplexy and pharmacological characteristics similar to those observed in humans having narcolepsy. Canine models of narcolepsy have been successfully used to predict or confirm the effects of a variety of compounds for their effects on wakefulness, and to screen for new compounds for use as treatments for human narcolepsy.

Methods for diagnosing disorders of excessive daytime sleepiness are well known to those skilled in the art. Therefore, those skilled in the art will be able to determine individuals having sleep disorders that could benefit from treatment with a PrRP receptor agonist that promotes wakefulness. In addition, such treatment can be beneficial to individuals absent such sleep disorders when alertness, awareness, consciousness, or lack of sleep are desired.

As used herein, the term "promoting wakefulness" refers to a decrease in sleepiness, tendency to fall asleep, or other symptoms of undesired or reduced alertness or consciousness compared with sleepiness, tendency to fall asleep, or other symptoms of undesired or reduced alertness or consciousness expected or observed without treatment. Promoting wakefulness refers to a decrease in any stage of sleep, including light sleep, deeper sleep characterized by the presence of high amplitude, low wave brain activity termed "slow wave sleep", and rapid eye movement (REM) sleep. A compound that promotes wakefulness can, for example, cause the animal to wake from sleep, prolong periods of wakefulness, prolong normal latency to sleep, restore normal sleep patterns following sleep deprivation, or enhance beneficial wake-like characteristics, such as alertness, responsiveness to stimuli, and energy.

In a further aspect, the invention provides a method of promoting sleep by administering an effective amount of a PrRP receptor antagonist. A mammal to be treated with a PrRP receptor antagonist is generally in need of sleep and could benefit physically or psychologically from increased quality or quantity of sleep. For example, a mammal having reduced or insufficient quality or quantity of sleep, such as reduced ability to fall asleep or stay asleep, having the tendency to awaken earlier than desired in the morning or having a sense of light or unrefreshing sleep can be considered to be in need of sleep.

A variety of physical and psychological conditions can lead to reduced quality or quantity of sleep in an individual. For example, insomnias such as adjustment sleep disorder and psychophysiologic insomnia; sleep disorders such as periodic limb movement disorder and restless legs syndrome; circadian rhythm disorders such as circadian rhythm sleep disorder and delayed sleep phase syndrome, are common causes of reduced quality or quantity of sleep. In addition, medical and psychiatric conditions, such as chronic pain and depression, can cause reduced quality or quantity of sleep. Further, medications used for treating a variety of conditions can cause reduced quality or quantity of sleep. Examples of such medications include β-blockers, corticosteroids, bronchodilators, selective serotonin reuptake inhibitors and thyroid hormone.

As used herein, the term "promoting sleep" refers to increasing the quality or quantity of sleep. For example, promoting sleep can increase the ability to fall asleep or stay sleep, increase the number of hours slept prior to waking and increasing the perceived depth or refreshing effect of sleep. A compound that promotes sleep can, for example, cause the animal to sleep, prolong periods of sleep, promote restful sleep, decrease sleep latency, or decrease unwanted wake-like characteristics, such as anxiety and hyperactivity.

Methods for diagnosing insomnia and related disorders of reduced sleep are well known to those skilled in the art. Therefore, those skilled in the art will be able to determine individuals having sleep disorders that could benefit from treatment with a PrRP receptor antagonist that promotes sleep. In addition, such treatment can be beneficial to individuals absent such sleep disorders when an increase in quality or quantity of sleep is desired.

A determination of whether wakefulness or sleep is promoted by treatment can be made, for example, by direct observation of behavioral or physiological properties of mammalian sleep, by self-reporting, or by various well-known methods, including electrophysiological methods. Such methods include, for example, examining electroencephalograph (EEG) activity amplitude and frequency patterns, referred to herein as "EEG measurement;" examining electromyogram activity, referred to herein as "EMG measurement;" and examining the amount of time during a measurement time period, in which a mammal is awake or exhibits a behavioral or physiological property characteristic of wakefulness, referred to herein as "wake time measurement."

PrRP was originally identified as a peptide having the physiological role of promoting the release of prolactin, a hormone involved in mammary development and lactation, from the anterior pituitary (Hinuma et al., Nature 393:272-276 (1998)).

As used herein, the term "PrRP receptor agonist" refers to a compound that selectively promotes or enhances normal signal transduction through the PrRP receptor. A PrRP receptor agonist can act by any agonistic mechanism, such as by binding a PrRP receptor at the normal PrRP binding site, thereby promoting PrRP receptor signaling. A PrRP receptor agonist can also act, for example, by potentiating the binding activity of PrRP or signaling activity of PrRP receptor. The methods of the invention can advantageously be used to identify a PrRP receptor agonist that acts through any agonistic mechanism. As described herein, an example of a PrRP receptor agonist is a "PrRP." A PrRP agonist can also be a "PrRP functional analog," as described below.

As used herein, the term "PrRP" refers to a peptide having identity with at least 5 residues of the native sequence of a mammalian prolactin-releasing peptide (PrRP), and which binds a "PrRP receptor" with an affinity (Kd) of about 10^-5M or less. A PrRP of the invention can thus have identity with at least 5, 6, 7, 8, 9, 10, 15, 20 or more contiguous or non-contiguous amino acid residues of a native PrRP. Preferably, a PrRP of the invention binds a PrRP receptor with a Kd of about 10^-6M or less, more preferably about 10^-7M or less, most preferably about 10^-8M or less, including about 10^-9M or less, such as 10^-10M or less.

Mature, native PrRP peptides exist in at least two forms, a 31 amino acid peptide (PrRP-31) and a 20 amino acid peptide (PrRP-20), which are amidated at the carboxy-terminus. PrRP-31 and PrRP-20 are derived from a longer preproprotein. The purification of PrRP-31 and PrRP-20 from bovine hypothalamus, the cloning of PrRP preproprotein from bovine, rat and human, the characterization of PrRP-31 and PrRP-20 as peptides having prolactin-releasing activity towards rat anterior pituitary cells in vitro, and the importance of the C-terminal amide for PrRP activity, are described in Hinuma et al., Nature 393:272-276 (1998).

The amino acid sequences of PrRP-31 from bovine, rat and human are as follows:

Bovine: SRAHQHSMEIRTPDINPAWYAGRGIRPVGRF (SEQ ID NO:13)

Rat: SRAHQHSMETRTPDINPAWYTGRGIRPVGRF (SEQ ID NO:14)

Human: SRTHRHSMEIRTPDINPAWYASRGIRPVGRF (SEQ ID NO:15)

The amino acid sequences of PrRP-20 from bovine, rat and human, which contain the C-terminal 20 amino acids of PrRP-31, are as follows:

Bovine: TPDINPAWYAGRGIRPVGRF (SEQ ID NO:16)

Rat: TPDINPAWYTGRGIRPVGRF (SEQ ID NO:17)

Human: TPDINPAWYASRGIRPVGRF (SEQ ID NO:18)

The term "PrRP" is intended to encompass PrRP-31 and PrRP-20 from bovine, rat and human, having the amino acid sequences shown above, as well as PrRP-31 and PrRP-20 from other mammalian species, including, for example, non-human primates, mouse, rabbit, porcine, ovine, canine and feline species. The sequences of PrRP from other mammalian species can be readily determined by those skilled in the art, for example either by purifying PrRP from hypothalamic extracts, or by cloning PrRP preproproteins, following the methods described in Hinuma et al., Nature 393:272-276 (1998). Because of the high degree of identity between bovine, rat and human sequences, it is expected that PrRP from other mammalian species will be substantially similar in structure and function to the known PrRP sequences.

The term "PrRP" is also intended to encompass peptides that are longer or shorter than PrRP-31 or PrRP-20, so long as they have identity with at least 5 residues of the native sequence of a mammalian prolactin-releasing peptide (PrRP), and can bind the PrRP receptor GPR10 with an affinity (Kd) of less than about 10^-5M. Thus, the term "PrRP" encompasses peptides that have one or several amino acid additions or deletions compared with the amino acid sequence of a PrRP-31 or PrRP-20. Those skilled in the art recognize that such modifications can be desirable in order to enhance the bioactivity, bioavailability or stability of the PrRP, or to facilitate its synthesis or purification.

The term "PrRP" is further intended to encompass peptides having identity with at least 5 residues of the native sequence of a mammalian prolactin-releasing peptide (PrRP), which bind a PrRP receptor with an affinity (Kd) of about 10^-5M or less, and which have one or several minor modifications to the native PrRP sequence. Contemplated modifications include chemical or enzymatic modifications (e.g. acylation, phosphorylation, glycosylation, etc.), and substitutions of one or several amino acids to a native PrRP sequence. Those skilled in the art recognize that such modifications can be desirable in order to enhance the bioactivity, bioavailability or stability of the PrRP, or to facilitate its synthesis or purification.

Contemplated amino acid substitutions to the native sequence of a PrRP include conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of an apolar amino acid with another apolar amino acid; replacement of a charged amino acid with a similarly charged amino acid, etc.). Those skilled in the art also recognize that nonconservative changes (e.g., replacement of an uncharged polar amino acid with an apolar amino acid; replacement of a charged amino acid with an uncharged polar amino acid, etc.) can be made without affecting the function of PrRP. Furthermore, non-linear variants of a PrRP sequence, including branched sequences and cyclic sequences, and variants that contain one or more D-amino acid residues in place of their L-amino acid counterparts, can be made without affecting the function of PrRP.

In particular, the term "PrRP" is intended to encompass peptides having minor modifications to the native PrRP sequence that serve to increase its penetration through the blood-brain barrier (BBB). For a review of strategies for increasing bioavailability of peptides and peptide drugs in the brain, and of methods for determining the permeability of peptides through the BBB using in vitro and in vivo assays, see Engleton et al., Peptides 9:1431-1439 (1997).

Strategies that have been successfully used to increase the permeability of other neuropeptides through the BBB are particularly contemplated. For example, modifying the opioid peptide analgesic Met-enkephalin with D-penicillamine at two positions, forming a disulfide bridge that conformationally constrains the peptide, dramatically increases its stability towards BBB endothelial cell proteases and its BBB permeability. Likewise, linking two enkephalin peptides, each containing a D-amino acid residue at the second position, with a hydrazide bridge, results in a metabolically stable peptide with improved brain penetration. Additionally, halogenation of an enkephalin peptide has been shown to increase its BBB permeability. Similar modifications to PrRP peptides are likewise expected to be advantageous.

Additional modifications to a PrRP peptide that can increase its BBB penetration include conjugating the peptide to a lipophilic moiety, such as a lipophilic amino acid or methyldihydropyridine. PrRP peptide can also be conjugated to a transporter, such as the monoclonal antibody OX26 which recognizes the transferrin receptor, or cationized albumin which utilizes the adsorptive mediated endocytosis pathway, so as to increase its BBB penetration.

Those skilled in the art can determine which residues and which regions of a native PrRP sequence are likely to be tolerant of modification and still retain the ability to bind PrRP receptor with high affinity. For example, amino acid substitutions, or chemical or enzymatic modifications, at residues that are less well conserved between species are more likely to be tolerated than substitutions at highly conserved residues. Accordingly, an alignment can be performed among PrRP sequences of various species to determine residues and regions in which modifications are likely to be tolerated.

Additional guidance for determining residues and regions of PrRP likely to be tolerant of modification is provided by studies of PrRP fragments and variants. For example, based on the observation that PrRP-20 has similar ability to transduce signals through the PrRP receptor as PrRP-31 (see, for example, Hinuma et al., Nature 393:272-276 (1998)), it is likely that the N-terminus of PrRP is highly tolerant of the modifications described herein.

In particular, as described in Roland et al., Endocrinology 140:5736-5745 (1999), a peptide designated PrRP(25-31), consisting of the C-terminal seven amino acids of PrRP (IRPVGRF, SEQ ID NO:23) binds GPR10 with an apparent affinity of 200 nM, compared with an affinity of about 1 nM for PrRP-31 or PrRP-20, and mobilizes calcium in CHOK1 cells transfected with GPR10. Thus, a peptide consisting of, or comprising, the amino acid sequence designated SEQ ID NO:23 is encompassed by the term "PrRP."

Alanine scanning mutagenesis of PrRP (25-31) indicates that variants with substitutions of Ile25, Pro27, Val28, or Phe31 retain the ability to bind GPR10 with an affinity of about 10^-6M. Thus, a PrRP can consist of, or comprise, the amino acid sequences XRPVGRF (SEQ ID NO:19), IRXVGRF (SEQ ID NO:20), IRPXGRF (SEQ ID NO:21), IRPVGRX (SEQ ID NO:22), where "X" is any amino acid, preferably a non-polar amino acid, more preferably alanine. Substitutions of Arg26 or Gly29 were shown to substantially reduce binding affinity of PrRP (25-31) for GPR10, and substitution of Arg30 completely eliminated binding. Substitution of either Arg26 or Arg30 with lysine or citrulline also completely eliminated binding. More generally, a PrRP peptide can be considered to consist of, or comprise, the amino acid sequence XRXXGRX, so long as it retains PrRP receptor binding activity.

In the modified PrRP sequences described above, the effect of amino acid substitutions on calcium signaling was commensurate with the effect on binding to GPR10 (see Roland et al., Endocrinology 140:5736-5745 (1999)). Accordingly, in view of the disclosure herein, it is predictable that a peptide considered-to be a "PrRP" by GPR10 binding criteria will also be functionally active in mediating G-protein coupled signaling through PrRP receptor, inhibiting AMPA mediated signaling in whole cell preparations, inhibiting oscillatory activity in RTN preparations, suppressing absence seizures in susceptible mammals, and preventing or treating neurological and psychiatric disorders in which PrRP-31 or PrRP-20 are effective. Thus, as described further below, a peptide having a modified PrRP sequence can be assayed by any of these functional criteria to confirm that it is a PrRP.

The PrRP peptides of the invention can be prepared in substantially purified form using either conventional peptide synthetic methods (see, for example, Roland et al., Endocrinology 140:5736-5745 (1999)), or using conventional biochemical purification methods, starting either from tissues containing PrRP or from recombinant sources (see, for example, Hinuma et al., Nature 393:272-276 (1998)).

As used herein, the term "PrRP receptor antagonist" refers to a compound that selectively inhibits or decreases normal signal transduction through the PrRP receptor. A PrRP receptor antagonist can act by any antagonistic mechanism, such as by binding a PrRP receptor or PrRP, thereby inhibiting binding between PrRP and PrRP receptor. A PrRP receptor antagonist can also act, for example, by inhibiting the binding activity of PrRP or signaling activity of PrRP receptor. For example, a PrRP receptor antagonist can act by altering the state of phosphorylation or glycosylation of PrRP receptor. The methods of the invention can advantageously be used to identify a PrRP receptor antagonist that acts through any antagonistic mechanism. A PrRP antagonist can be, for example, a "PrRP functional analog," as described below.

As used herein, the term "PrRP functional analog" refers to a molecule that binds the PrRP receptor GPR10 with an affinity (Kd) of about 10^-5 M or less, and which is not encompassed within the definition of a "PrRP," as set forth above. Preferably, a PrRP functional analog will bind a PrRP receptor with a Kd of about 10^-6M or less, more preferably about 10^-7M or less, most preferably about 10^-8M or less, including about 10^-9M or less, such as about 10^-10M or less.

The PrRP functional analogs of the invention can act as PrRP receptor agonists, and thus be able to mediate the same biochemical and pharmacological effects (e.g. signal transduction through the PrRP receptor, reduction of AMPA receptor activity, suppression of absence seizures in mammals) as PrRP. A PrRP functional analog identified by the methods described herein can alternatively act as a PrRP receptor antagonist, and thus inhibit signaling through GPR10, prevent the suppression of AMPA receptor mediated activity, or both. Such antagonists can advantageously be used in therapeutic applications where a reduction in PrRP receptor signaling is desired, including in the treatment of sleep and attention disorders. PrRP functional analogs of the invention, which are themselves not appropriate for therapeutic use, can advantageously be used to optimize the design of effective therapeutic compounds, or used in the screening methods described herein as competitors.

A PrRP functional analog can be a naturally occurring macromolecule, such as a peptide, nucleic acid, carbohydrate, lipid, or any combination thereof. A PrRP functional analog also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic or inorganic molecule prepared partly or completely by synthetic chemistry methods. A PrRP functional analog can be identified starting either by rational design based on the corresponding peptide, by functional screening assays, or by a combination of these methods.

PrRP functional analogs include peptidomimetics of PrRP, such as peptidomimetics of a peptide containing, or consisting of, the amino acid sequence set forth as SEQ ID NO:23. As used herein, the term "peptidomimetic" refers to a non-peptide agent that is a topological analog of the corresponding peptide. Those skilled in the art understand that the identified ability of PrRP-31, PrRP-20, PrRP(25-31) and of certain single amino acid variants of PrRP(25-31) to bind PrRP receptor with-high affinity, provides sufficient structural and functional information to rationally design peptidomimetics of PrRP.

Such a peptidomimetic can, for example, retain some or all of the functional groups of the amino acids shown to be functionally important in the C-terminus of PrRP (such as the 3-guanylpropyl radical of Arg26 and Arg30, the hydrogen of Gly29, etc.). A peptidomimetic of PrRP can also, for example, consist partially or completely of a non-peptide backbone used in the art in the design of other peptidomimetics, such as a glucose scaffold, a pyrrolidinone scaffold, a steroidal scaffold, a benzodiazepine scaffold, or the like.

Methods of rationally designing peptidomimetics of peptides, including neuropeptides, are known in the art. For example, the rational design of three peptidomimetics based on the sulfated 8-mer peptide CCK26-33, and of two peptidomimetics based on the 11-mer peptide Substance P, and related peptidomimetic design principles, are described in Horwell, Trends Biotechnol. 13:132-134 (1995).

Individual, rationally designed peptidomimetics of PrRP peptides can be assayed for their ability to bind the PrRP receptor, or to induce signaling through the PrRP receptor, or both, using one or more of the assays described herein. Similarly, a plurality of peptidomimetic compounds, such as variants of a peptidomimetic lead compound, or a plurality of other compounds, can be assayed simultaneously or sequentially using the binding, signaling and pharmacological assays described herein.

In methods of controlling absence seizures, promoting wakefulness and sleep, and for certain other therapeutic applications, a PrRP functional analog can be used. In comparison to a PrRP peptide, a PrRP functional analog can be more stable, more active, or have higher inherent ability to penetrate the blood-brain barrier than a PrRP.

A candidate compound can be assayed to determine whether it is a PrRP agonist or antagonist, either by a signaling assay, a binding assay, or both. The number of different compounds to screen in a particular assay can be determined by those skilled in the art, and can be 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds. For certain applications, such as when a library of random compounds is to be screened, and for automated procedures, it may be desirable to screen 10³or more compounds, such as 10⁵or more compounds, including 10⁷or more compounds.

Methods for producing large libraries of chemical compounds, including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.

In one embodiment, a signaling assay can be performed to determine whether a candidate compound is a PrRP receptor agonist or antagonist. In such an assay, a PrRP receptor is contacted with one or more candidate compounds under conditions wherein the PrRP receptor produces a predetermined signal in response to a PrRP agonist, such as PrRP. In response to PrRP receptor activation, a predetermined signal can be an increase or a decrease from an unstimulated baseline signal. An example of a predetermined signal which increases from an unstimulated baseline signal is a detected second messenger molecule that is produced in response to PrRP receptor activation. An example of a predetermined signal which decreases from an unstimulated baseline signal is a detected second messenger molecule that is hydrolyzed in response to PrRP receptor activation. The production of a predetermined signal in response to PrRP receptor agonist binding to PrRP receptor can therefore be an increase in a predetermined signal which positively correlates with PrRP receptor activity, or a decrease in a predetermined signal which negatively correlates with PrRP receptor activity. Using a signaling assay of the invention, a PrRP receptor agonist is identified that promotes production of a predetermined signal, whether the agonist promotes an increase in a predetermined signal that positively correlates with PrRP receptor activity, or a decrease in a predetermined signal that negatively correlates with PrRP receptor activity.

Similarly, a signaling assay can be performed to determine whether a candidate compound in a PrRP receptor antagonist. In such an assay, a PrRP receptor is contacted with one or more candidate compounds under conditions wherein the PrRP receptor produces a predetermined signal in response to a PrRP agonist, such as PrRP, and a compound is identified that reduces production of the predetermined signal. The candidate compound can be tested at a range of concentrations to establish the concentration where half-maximal signaling occurs; such a concentration is generally similar to the dissociation constant (Kd) for PrRP receptor binding.

As used herein, the term "PrRP receptor," is intended to refer to a mammalian seven-transmembrane-domain G-protein coupled receptor, variously designated in the art "GPR10" (Marchese et al., Genomics 29:335-344 (1995)), "hGR3" (Hinuma et al., Nature 393:272-276 (1998)) or "UHR-1" (Welch et al., Biochem. Biophys. Res. Commun. 209:606-613 (1995)). A "PrRP receptor" can have minor modifications to the native mammalian sequence, so long as the minor modifications do not significantly alter its ability to bind PrRP, interact with AMPA receptor associated molecules, signal through a G-protein coupled signal transduction pathway, or modulate AMPA receptor signaling, depending on the particular application of the PrRP receptor in the methods of the invention.

The PrRP receptor to be contacted in the methods of the invention can be naturally expressed in a tissue, cell or extract. Alternatively, where it is desired to increase the PrRP receptor concentration, or to express PrRP receptor in host cells where it is not normally expressed, including mammalian, yeast and bacterial cells, the PrRP receptor can be recombinantly expressed. Methods of recombinantly expressing PrRP receptor, either transiently or stably, in a variety of host cells, are well known in the art (see, for example, Hinuma et al., Nature 393:272-276 (1998) and Roland et al., Endocrinology 140:5736-5745 (1999)).

As used herein, the term "predetermined signal" refers to a readout, detectable by any analytical means, that is a qualitative or quantitative indication of activation of G-protein-dependent signal transduction through PrRP receptor. The term "G-protein" refers to a class of heterotrimeric GPT binding proteins, with subunits designated Gα, Gβ and Gγ, that couple to seven-transmembrane cell surface receptors to transduce a variety of extracellular stimuli, including light, neurotransmitters, hormones and odorants to various intracellular effector proteins. G proteins are present in both eukaryotic and prokaryotic organisms, including mammals, other vertebrates, Drosophila and yeast.

As described in Hinuma et al., Nature 393:272-276 (1998), contacting PrRP receptor with PrRP leads to activation of arachidonic acid metabolite release in mammalian cells recombinantly expressing PrRP receptor. Therefore, an exemplary predetermined signal that is a qualitative or quantitative indication of activation of G protein-dependent signal transduction through PrRP receptor is arachadonic acid metabolite release. Similarly, as described in Roland et al., Endocrinology 140:5736-5745 (1999), contacting PrRP receptor with PrRP leads to calcium mobilization in mammalian cells recombinantly expressing PrRP receptor, which can be measured, for example, using the calcium indicator fluo-3 and a fluorescence monitoring system.

If desired, a predetermined signal other than arachadonic acid metabolite release or Ca²⁺ influx can be used as the readout in the methods of the invention. The specificity of a G-protein for cell-surface receptors is determined by the C-terminal five amino acids of the Gα subunit. The nucleotide sequences and signal transduction pathways of different classes and subclasses of Gα subunits in a variety of eukaryotic and prokaryotic organisms are well known in the art. Thus, any convenient G-protein mediated signal transduction pathway can be assayed by preparing a chimeric Gα containing the C-terminal residues of a Gα that couples to PrRP receptor, such as Gαq, with the remainder of the protein corresponding to a Gα that couples to the signal transduction pathway it is desired to assay.

Methods of recombinantly expressing chimeric Gα proteins, and their use in G-protein signaling assays, are known in the art and are described, for example, in, and Saito et al., Nature 400:265-269 (1999), and Coward et al., Anal. Biochem. 270:2424-248 (1999)).

Signaling through G proteins can lead to increased or decreased production or liberation of second messengers, including, for example, arachidonic acid, acetylcholine, diacylglycerol, cGMP, cAMP, inositol phosphate and ions; altered cell membrane potential; GPT hydrolysis; influx or efflux of amino acids; increased or decreased phosphorylation of intracellular proteins; or activation of transcription. Thus, by using a chimeric Gα subunit that binds PrRP receptor and couples to a desired signal transduction pathway in the methods of the invention, those skilled in the art can assay any convenient G-protein mediated predetermined signal in response to a PrRP receptor agonist or antagonist.

Various assays, including high throughput automated screening assays, to identify alterations in G-protein coupled signal transduction pathways are well known in the art. Various screening assay that measure Ca⁺⁺, cAMP, voltage changes and gene expression are reviewed, for example, in Gonzalez et al., Curr. Opin. in Biotech. 9:624-631 (1998); Jayawickreme et al., Curr. Opin. Biotech. 8:629-634 (1997); and Coward et al., Anal. Biochem. 270:2424-248 (1999). Yeast cell-based bioassays for high-throughput screening of drug targets for G-protein coupled receptors are described, for example, in Pausch, Trends in Biotech. 15:487-494 (1997). A variety of cell-based expression systems, including bacterial, yeast, baculovirus/insect systems and mammalian cells, useful for detecting G-protein coupled receptor agonists and antagonists are reviewed, for example, in Tate et al., Trends in Biotech. 14:426-430 (1996).

Assays to detect and measure G-protein-coupled signal transduction can involve first contacting the isolated cell or membrane with a detectable indicator. A detectable indicator can be any molecule that exhibits a detectable difference in a physical or chemical property in the presence of the substance being measured, such as a color change. Calcium indicators, pH indicators, and metal ion indicators, and assays for using these indicators to detect and measure selected signal transduction pathways are described, for example, in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Sets 20-23 and 25 (1992-94). For example, calcium indicators and their use are well known in the art, and include compounds like Fluo-3 AM, Fura-2, Indo-1, FURA RED, CALCIUM GREEN, CALCIUM ORANGE, CALCIUM CRIMSON, BTC, OREGON GREEN BAPTA, which are available from Molecular Probes, Inc., Eugene Oreg., and described, for example, in U.S. Pat. Nos. 5,453,517, 5,501,980 and 4,849,362.

Assays to determine changes in gene expression in response to a PrRP receptor agonist or antagonist can involve first transducing cells with a promoter-reporter nucleic acid construct such that a protein such as β-lactamase, luciferase, green fluorescent protein or β-galactosidase will be expressed in response to contacting PrRP receptor with a PrRP receptor agonist or antagonist. Such assays and reporter systems are well known in the art.

An assay to determine whether a candidate compound is a PrRP receptor agonist or antagonist, is performed under conditions in which contacting the receptor with a known PrRP agonist, such as a PrRP, including PrRP-31 or PrRP-20, would produce a predetermined signal. If desired, the assay can be performed in the presence of a known PrRP agonist, such as a PrRP. Preferably, the PrRP concentration will be within 10-fold of the EC₅₀. Thus, an agonist that competes with PrRP for signaling through the PrRP receptor, or indirectly potentiates the signaling activity of PrRP, can be readily identified. Likewise, an antagonist that prevents PrRP from binding the PrRP receptor, or indirectly decreases the signaling activity of PrRP, can also be identified.

As described in "this patent", functional interaction of PrRP with GPR10 results in the association of GPR10 through its C-terminus with AMPA receptor associated molecules. Thus, a further signaling assay for identifying a PrRP agonist or antagonist consists of contacting a PrRP receptor with a candidate compound under conditions wherein PrRP promotes interaction of PrRP receptor with an AMPA receptor associated protein, and determining the ability of the candidate compound to promote the interaction of the PrRP receptor with the AMPA receptor associated protein. A candidate compound that promotes the interaction of PrRP receptor with an AMPA receptor associated protein is characterized as a PrRP receptor agonist. In contrast, a candidate compound that reduces the interaction of PrRP receptor with an AMPA receptor associated protein is characterized as a PrRP receptor antagonist.

Exemplary AMPA receptor associated molecules include PICK1 (see Xia et al., Neuron 22:179-187 (1999)), GRIP1 (Dong et al., J. Neurosci. 19:6930-6941 (1999)), and GRIP2/ABP (Dong et al., J. Neurosci. 19:6930-6941 (1999); Srivista et al., Neuron 21:581-591 (1998)), which are PDZ domain containing proteins, and other proteins that similarly interact with the GluR2 or GluR3 subunits of AMPA receptors.

Methods of determining the interaction between PrRP receptor and an AMPA receptor associated protein, and suitable compositions for practicing the methods, are described in "this patent". For example, a cell, such as a mammalian, yeast or bacterial cell, can be cotransfected with a nucleic acid expression construct directing the expression of PrRP receptor, and a nucleic acid molecule expression construct directing the expression of AMPA receptor associated protein, and the cell contacted with a candidate compound. Interaction between the PrRP receptor and AMPA receptor associated protein following such contacting can be determined, for example, by co-immunoprecipitation of the two proteins, or by intracellular or surface clustering of the two proteins. Nucleic acid expression constructs and suitable host cells for expressing PrRP receptor and AMPA receptor associated proteins, and immunological reagents and methods suitable for detecting such interactions, are known in the art.

A candidate compound can alternatively or additionally be assayed to determine whether it is a PrRP receptor agonist or antagonist by a PrRP receptor binding assay. If desired, a binding assay can be followed by a signaling assay, to determine whether the identified compound is a PrRP receptor agonist or antagonist. Receptor binding assays, including high-throughput automated binding assays, are well known in the art, and any suitable direct or competitive binding assay can be used. Exemplary high-throughput receptor binding assays are described, for example, in Mellentin-Micelotti et al., Anal. Biochem. 272: P182-190 (1999); Zuck et al., Proc. Natl. Acad. Sci. USA 96:11122-11127 (1999); and Zhang et al., Anal. Biochem. 268;134-142 (1999). The assay format can employ a cell, cell membrane, or artificial membrane system, so long as the PrRP receptor is in a suitable conformation for binding PrRP with a similarly affinity and specificity as a PrRP receptor expressed on the surface of a mammalian cell.

Contemplated binding assays can involve detectably labeling a candidate compound, or competing an unlabeled candidate compound with a detectably labeled PrRP agonist, such as a PrRP. A detectable label can be, for example, a radioisotope, fluorochrome, ferromagnetic substance, or luminescent substance. Exemplary radiolabels useful for labeling compounds include ¹²⁵I, ¹⁴C and ³H. Methods of detectably labeling organic molecules, either by incorporating labeled amino acids into the compound during synthesis, or by derivatizing the compound after synthesis, are known in the art.

In the binding and signaling assays described above, appropriate conditions for determining whether a compound is a PrRP agonist or antagonist are those in which a control PrRP exhibits the binding or signaling property. The control assay can be performed before, after or simultaneously with the test assay.

The invention also provides methods of identifying compounds that modulate AMPA receptor signaling, including compounds that suppress AMPA receptor signaling and compounds that enhance AMPA receptor signaling. Such compounds can be used, for example, as therapeutic compounds for controlling absence seizures, promoting wakefulness, and promoting sleep, as well as in the prevention and treatment of conditions associated with tissues in which GPR10 is expressed. Such compounds can also be used, for example, in the design and development of compounds which themselves can be used as therapeutics, or for further analysis of biochemical pathways.

The method consists of providing one or more compounds that are PrRP receptor agonists or antagonists and determining the ability of the compound to modulate AMPA receptor signaling. The one or more compounds that are PrRP receptor agonists or antagonists can be identified, isolated or prepared by the methods and criteria set forth above.

Assays for determining AMPA receptor signaling can either directly measure AMPA receptor electrophysiological activity in a cell or tissue, or measure a biochemical or physiological property that is correlated with AMPA receptor activity. Appropriate assays and conditions for determining whether a compound modulates AMPA receptor signaling are those in which a control PrRP modulates AMPA receptor signaling. The control assay can be performed before, after or simultaneously with the test assay, depending on the particular assay. Such assays are known in the art or described herein, and include both manual and high-throughput automated assays.

A method of determining whether a PrRP receptor agonist or antagonist modulates AMPA receptor electrophysiological activity can involve determining AMPA receptor-mediated oscillatory activity in a tissue, such as a neural tissue, that expresses both PrRP receptors and AMPA receptors. "This patent" describes exemplary conditions for determining AMPA receptor-driven oscillatory activity in a thalamic preparation. Application of PrRP reduced AMPA receptor mediated oscillatory activity, in a dose-dependent manner. Accordingly, an assay of thalamic oscillatory activity can be used to determine whether a compound modulates AMPA receptor signaling.

A further method of determining whether a PrRP receptor agonist or antagonist modulates AMPA receptor electrophysiological activity can involve an assay of the electrophysiological properties of a single cell or cell population which normally expresses (e.g. RTN neurons), or which recombinantly expresses, functional PrRP receptors and AMPA receptors. Methods of transiently or stably transfecting cells with AMPA receptors are well known in the art and are described, for example, in Hall et al., J. Neurochem. 68:625-630 (1997), and in Hennegriff et al., J. Neurochem. 68:2424-2434 (1997).

"This patent", and Smith et al., J. Neuroscience 20:2073-2085 (2000), describe exemplary conditions for determining AMPA receptor mediated electrophysiological recordings from whole cells. In brief, the method involves detecting AMPA receptor mediated currents using whole cell patch clamp recordings in the presence of an AMPA agonist. The modulatory effect of a test compound on the AMPA receptor mediated currents can thus be determined. Such assays can be performed in the presence of a drug such as cyclothiazide to reduce AMPA receptor densensitization.

Alternatively, or additionally, a method of determining whether a PrRP agonist or antagonist modulates AMPA receptor signaling activity can involve an assay of AMPA receptor-mediated second messenger responses in cells expressing functional PrRP receptors and AMPA receptors. Such assays are advantageous in that they are readily amenable to automation, using methods known in the art, allowing rapid and high-throughput screening of compounds.

"This patent" describes exemplary conditions for determining AMPA receptor mediated calcium ion or sodium ion influx into cells in response to a compound that modulates AMPA receptor signaling. In brief, the method involves detecting AMPA receptor mediated ion influx using fluorescent ion indicators and either microscopic visualization, or an automated fluorometric imaging plate reader (FLIPR). The modulatory effect of a test compound on AMPA receptor mediated ion influx can thus be determined.

The invention also provides methods of identifying compounds for controlling absence seizures. The method consists of providing a compound that is a PrRP agonist, and determining the ability of the compound to control absence seizures in a mammal. Optionally, the compound can be a compound determined to suppress AMPA receptor mediated signaling by any of the assays described herein.

Assays for determining whether a compound controls absence seizures in a mammal are known in the art. For example, as described in "this patent", absence seizure activity can be determined in a mammalian model of absence epilepsy, the GAERS, in which spontaneous spike-and-wake discharges are evidenced by EEG recordings. Administration of PrRP decreased seizure activity in the GAERS, in a dose-dependent manner. Accordingly, an in vivo assay in a mammal susceptible to absence seizures, including a rodent, non-human primate, or human, can be used to identify a compound for controlling absence seizures.

"This patent" describes exemplary conditions for determining the ability of a PrRP receptor agonist to promote wakefulness in mammals. In brief, the methods involve obtaining EEG and EMG patterns and observing behavioral properties correlated with mammalian wakefulness or sleep, such as activity. An EEG pattern characterized by high amplitude waves associated with deep sleep was observed prior to PrRP treatment. Upon PrRP treatment, this EEG pattern was altered to an EEG pattern characterized by lower amplitude waves associated with increased wakefulness. The silent EMG pattern observed prior to PrRP treatment was characteristic of REM sleep, while the EMG pattern observed upon PrRP treatment was characteristic of increased wakefulness. The effect of a PrRP receptor agonist or antagonist on a wakefulness or sleep state of a mammal can thus be determined using the experimental system described in "this patent".

The invention further provides methods of screening for compounds for promoting wakefulness. The method consists of providing a compound that is a PrRP receptor agonist and determining the ability of the compound to promote wakefulness in a mammal. Optionally, the compound can be a compound determined to suppress AMPA receptor mediated signaling in any of the assays described herein.

In addition, the invention provides methods of screening for compounds for promoting sleep. The method consists of providing a compound that is a PrRP receptor antagonist and determining the ability of the compound to promote sleep in a mammal.

Assays for determining whether a compound promotes wakefulness or sleep in a mammal are known in the art. For example, as described in "this patent", wakefulness and sleep can be determined in a strain of normal rat. Administration of PrRP increased wakefulness, as evidenced by cortical EEG (ECoG), EMG and wake time measurements, in a dose-dependent manner. Accordingly, an in vivo assay in any mammal, including a rodent, canine, horse, non-human primate, or human, can be used to identify a compound for promoting wakefulness or sleep.

A candidate compound can be tested for its effects on one or more behavioral and physiological properties correlated with mammalian sleep or wake states. Behavioral properties correlated with mammalian sleep or wake states include, for example, activity, sleep latency, and arousal threshold. Activity includes all behavioral activities normally exhibited by a mammal, such as movement, grooming, eating and the like. In humans, an exemplary activity that can be useful for determining quality of sleep is major body repositioning, which can be assessed as rate of major body position changes per hour. Activity can be evaluated throughout a normal wake period or throughout a normal sleep period, or both, or evaluated for only part of a normal wake or sleep period, such as for at least 10 minutes, 30 minutes, 1, 2, 4, 6, 8 or 12 hours. Once activity during a normal sleep period or normal wake period is established, those skilled in the art can readily evaluate whether a candidate compund increases or decreases intensity of activity or alters the pattern of activity during all or part of that period.

For certain applications, it will be preferable to evaluate activity following sleep deprivation. For example, a compound that promotes wakefulness or sleep can be examined in a sleep-deprived mammal. Sleep deprivation is generally performed for a sufficient period of time during a normal sleep period to result in a detectable decrease in activity, increase in sleep or increase in intensity of sleep during the subsequent period, also known as a rebound effect. Any method appropriate for the particular mammal can be used to deprive an animal of sleep. In certain mammals, such as humans, it can be preferable to cause sleep-deprivation by using noise or other stimulation for short or long periods. In particular, slow wave sleep can be reduced or interrupted by stimulating a mammal when EEG or other measurements indicate the beginning of a slow wave sleep stage.

Various manual and automated methods can be used to evaluate intensity and patterns of activity. For example, activity can be detected visually, either by direct observation, by time lapse photography and by use of an activity detector, such as an actigraph, which can be conveniently used to detect movement and nonmovement in humans. For humans, sleep logs, diaries, self-administered questionnaires and symptom checklists can be useful for determining activity and sleep quality and quantity.

Another behavioral property correlated with mammalian sleep or wake states is sleep latency, which refers to the number of minutes before the onset of a measurable sleep cycle. Once normal latency to sleep, with or without sleep deprivation, is established for a particular mammal, one skilled in the art can evaluate whether a candidate compound increases or decreases this property. A further behavioral property useful for assessing human wake and sleep states is sleep efficiency, which is the percentage of time spent in bed versus the time spent asleep. Once normal sleep efficiency, with or without sleep deprivation, is established for a particular individual, one skilled in the art can evaluate whether a candidate compound increases or decreases this property.

Arousal threshold refers to the amount of stimulation required to elicit a behavioral response. Any reproducible stimulus can be used to evaluate arousal threshold, for example, vibratory stimulus, noise, electrical stimulation, heat, light and the like. Arousal threshold can be assessed by determining activity, such as by determining the number per hour of measurable, but brief, changes during sleep to waking brain wave activity. Mammals that are in a state of wakefulness will exhibit a behavioral response at a lower level of stimulation than mammals that are in a sleep state. Further, an animal that is deeply asleep will exhibit an increased arousal threshold compared to an animal that is less deeply asleep. Therefore, arousal threshold is a measurement of sleep versus wakefulness, as well as intensity of sleep. Once normal arousal threshold associated with sleep and wakefulness are established for a particular mammal, those skilled in the art can readily evaluate whether a candidate compound increases or decreases this property.

Physiological properties correlated with mammalian sleep or wake states include frequency, amplitude and type of electrophysiological signals, heart rate, muscle tone, eye movement and the like. Electrophysiological measurements can be used to determine a stage of sleep experienced by a mammal, as well as the duration of a stage of sleep, and a change in sleep stage. Sleep stages are variations in states of consciousness and include, for example, light sleep, deeper sleep, such as slow wave sleep, and REM sleep. Correlative electrophysiological measurements characteristic of such sleep stages are well known to those skilled in the art.

Use of electrophysiological methods for determining sleep stage and correlation of various phases and states of sleep and arousal are described, for example, in Timo-Iaria et al., Physiology and Behavior, 5:1057-1062, (1970) and Vanderwolf et al. The Behavioral and Brain Sciences, 4:459-514 (1981). Brain wave activity associated with sleep or wakefulness can be determined, for example, by electroencephalogram (EEG) measurement. In particular, forebrain electroencephalograph activity amplitude or frequency patterns can be used to determine a state of wakefulness or sleep in a mammal, including a human. For example, during non-rapid eye movement (non-REM) sleep, cortical EEG (ECoG) exhibits predominant large amplitude, slow-wave activity (<1 Hz) while low-amplitude, high-frequency fluctuations are typically observed during most periods of alert waking and REM sleep. Light sleep can be characterized by EEG wave patterns termed sleep spindles, which are increases in wave frequency, and K complexes, which are increases in wave amplitude. In contrast, deep sleep can be characterized by EEG wave patterns containing slow, high amplitude brain waves. Therefore, electrophysiological measurements, such as EEG measurement, in particular ECoG measurement, can be used to determine a state of wakefulness or sleep in a mammal.

REM sleep, which is characterized by sudden and substantial loss in muscle tone and an increase in rapid eye movement, can be distinguished from other sleep stages using various electrophysiological measurements. For example, REM sleep can be distinguished from other stages of sleep by measurement of muscle tone by electromyography (EMG) and measurement of eye movement by electro-oculogram (EOG). "This patent" describes the use of EMG from dorsal neck muscles to monitor sleep in rats. Methods of using EMG for monitoring sleep in individuals, such as chin EMG, as well as EOG, are well known to those skilled in the art. Therefore, the effect of a compound that promotes wakefulness or sleep on specific stages of sleep, such as an increase or decrease in slow wave sleep or REM sleep, can be determined. A combination of two or more electrophysiological measurements can be used to determine a sleep stage, duration of sleep stage or change in sleep stage in a mammal.

Methods for evaluating sleep in a mammal are useful for both diagnosing a variety of sleep disorders to determine if an individual is a candidate for treatment with a PrRP receptor agonist or antagonist, as well as to evaluate an individual's response to administration of a PrRP receptor agonist or antagonist. Methods for evaluating sleep can involve continuous and simultaneous monitoring of various behavioral and physiological parameters of sleep. Such sleep evaluations include nighttime sleep studies, such as a polysomnogram, and daytime sleep studies, such as a Multiple Sleep Latency Test. Such sleep studies can be used to assess the quality and quantity of sleep by determining types of sleep stages experienced, duration of sleep, arousal threshold, sleep latency, activity, and other measurements, if desired. A sleep study can include, for example, electrophysiologic methods, such as measurements from an electroencephalogram (EEG), electro-oculogram (EOG) or electromyogram (EMG). In addition to these electrophysiologic methods, other measurements and conditions of a mammal can be monitored, for example, electrocardiogram (ECG), airflow, ventilation and respiratory effort, transcutaneous monitoring or end tidal gas analysis, extremity muscle activity, motor activity movement, gastroesophageal reflux, continuous blood pressure monitoring, snoring, body positions, amount of REM sleep, determination of the latency to the first REM episode, and the like. Those skilled in the art will know how to review, interpret and report the findings of such monitoring.

Following administrating of a candidate compound to a mammal, wake time, EEG and EMG measurements and any of the behavior or physiological properties correlated with mammalian wake or sleep states described above can be evaluated, and a determination made as to whether the compound alters, such as increases or decreases, the measurement or property compared to a baseline or established value for the measurement or property in an untreated control.

Additionally, a candidate compound can be tested for its effects on one or more additional behavioral or physiological properties in order to determine its most effective application in therapy. For example, it may be desirable to determine whether a compound that promotes wakefulness does so without significantly altering sleep latency when the effect of the compound wears off. It may also be desirable to determine whether the compound that promotes wakefulness does so without a compensatory sleep rebound effect. It can be further be desirable to determine whether the compound that promotes wakefulness effects other physiological or psychological properties or behaviors such as locomotor activity, anxiety, blood pressure and heart rate.

The methods of the invention for screening for a compound for promoting wakefulness are useful for identifying a PrRP receptor agonist that promotes wakefulness. Therefore, the invention provides a method of promoting wakefulness in an animal by administering to the mammal an effective amount of a PrRP receptor agonist.

An amount of a PrRP agonist effective to promote wakefulness is an amount effective to reduce a determined parameter (for example, amount of sleep, sleepiness, tendency to fall asleep, slow wave sleep) or increase a determined parameter (for example, wake time, sleep latency, activity) by at least 10%. Preferably, the determined parameter will be reduced by at least 20%, more preferably at least 50%, such as at least 80%, in at least some treated mammals. Accordingly, a treatment that promotes wakefulness will be useful in improving the quality of life or obtaining the desired level of wakefulness in the treated mammals. Further description of effective amounts, formulations and routes of administration of PrRP agonists useful in the methods of the invention is provided below.

The methods of the invention for screening for a compound for promoting sleep are useful for identifying a PrRP receptor antagonist that promotes sleep. Therefore, the invention provides a method of promoting sleep in an animal. The method consists of administering to the mammal an effective amount of a PrRP receptor antagonist.

An amount of a PrRP antagonist effective to promote sleep is an amount effective to increase the determined parameter (for example, sleep, sleepiness, tendency to fall asleep, slow wave sleep, arousal threshold) or decrease the determined parameter (for example, wake time, activity, sleep latency) by at least 10%. Preferably, the determined parameter will be reduced by at least 20%, more preferably at least 50%, such as at least 80%, in at least some treated mammals. Accordingly, a treatment that promotes sleep will be useful in improving the quality of life in the treated mammals. Further description of effective amounts, formulations and routes of administration of the PrRP antagonists useful in the methods of the invention is provided below.

It is expected that the PrRP receptor agonists will have beneficial activities apart from, or in addition to, controlling absence seizures and promoting wakefulness. It is similarly expected that the PrRP receptor antagonists will have beneficial activities apart from, or in addition to, promoting sleep. As described herein, high levels of GPR10 expression have been observed in a number of discrete locations in the brain and peripheral tissues. In particular, GPR10 is expressed at high levels in the GABAergic neurons of the RTN. The GABAergic neurons of the RTN change their firing patterns in response to sleep and wake states. During periods of EEG-synchronized, deep sleep, RTN neurons generate rhythmic, high-frequency bursts of action potentials, while during waking and REM sleep, these neurons generate sequences of tonic action potential activity (for a review, see McCormick et al., Annu. Rev. Neurosci., 20:185-215 (1997)). GPR10 is expressed in other areas of the brain known to be involved in regulating sleep and attention, for example, preoptic and hypothalamic areas of the brain, such as the tuberomammillary nucleus, as well as in the locus coeruleus. Accordingly, it is contemplated that PrRP receptor agonist and antagonists, will be effective in preventing or ameliorating sleep disorders and attention disorders by modulating signaling through the GABAergic neurons of the RTN. Attention disorders are well known in the art and include, for example, attention deficit hyperactivity disorder, affective disorders, and disorders of memory.

A variety of sleep disorders are also well known in the art and are described, for example, in Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (1994), published by the American Psychiatric Association. The most common sleep disorder is primary insomnia, or a difficulty in initiating or maintaining sleep, which affects a large percentage of the population at some point in their lives. Other common sleep disorders include hypersomnia, or excessive daytime sleepiness, narcolepsy, which is characterized by sudden and irresistible bouts of sleep, and sleep apnea, which is a temporary cessation of breathing during sleep.

As described herein, GPR10 is also expressed in the Area Postrema (AP), Bed nucleus stria terminalis (BST), Central nucleus amygdala (CeA), parabrachial nucleus (PB), dorsal raphe, caudal (DRC), hypothalamic nucleus (Hypo), hypothalamic paraventricular nucleus (PVN), locus coeruleus (LC), lateral hypothalamus nucleus (LH), lateral preoptic nucleus (LPO), median preoptic nucleus (MnPO), MPA (medial preoptic area), MPO (medial preoptic nucleus), nucleus of the solitary tract (NTS), periventricular nucleus (Pe), suprachiasmatic nucleus (Sch), supraoptic nucleus (SO), Superior colliculus (SC), and Shell, nucleus accumbens (SNAc), dorsomedial hypothalamic nucleus (DMH), ventromedial hypothalamus (VMH), ventral tuberomamillary nucleus (VTM), ventromedial preoptic nucleus (VMPO) of the brain, as well as in peripheral tissues including the Adrenal medulla (AdM), uterus. Accordingly, it is contemplated that PrRP receptor agonists and antagonists will be effective in preventing, ameliorating or modulating conditions associated with these regions of the brain and periphery, including those shown in Table 1, below.

TABLE 1

Therapeutic Potential of PrRP

Therapeutic Potential	GPR10 Localization

Bulimia	LH, VLH, VMH, DMH,
	Arc, SNAc,
	NTS, AP
Anorexia	LH, VLH, VMH, DMH,
	Arc, SNAc,
	NTS, AP
Obesity	LH, VLH, VMH, DMH,
	Arc, SNAc,
	NTS, AP
Chronic pain states	PB, NTS
Stress-induced anorexia	Hypo, BST, CeA
Stress-induced hypertensive crisis	Lateral septum,
	BST, CeA, PVN, LC,
	SNAc
Anxiety	Lateral septum,
	BST, CeA, PVN, LC,
	SNAc
Excessive fear response	Lateral septum,
	BST, CeA, PVN, LC,
	SNAc
Excessive stress	Lateral septum,
	BST, CeA, PVN, LC,
	SNAc
Posttraumatic Stress disorder	BST, CeA, Hypo, AP
Nicotine induced cardiac arrhythmias	NTS, AP
Nicotine induced coronary spasms	NTS, AP
Pheochromocytoma	AdM
Insomnia	RTN, MnPO, MPA,
	VMPO, LPO, VTM,
	LC, DRC, SCh
Hypersomnia syndrome	RTN, MnPO, MPA,
	VMPO, LPO, VTM,
	LC, DRC, SCh
Narcolepsy	RTN, MnPO, MPA,
	VMPO, LPO, VTM,
	LC, DRC, SCh
Excessive somnolence (need for	RTN, MnPO, MPA,
alertness)	VMPO, LPO, VTM,
	LC, DRC, Sch
Petit mal (absence) seizure	RTN
Visual processing and attention	SC
deficits
Drug addiction	SNAc
Inducing labor	Uterus
Birth control	Uterus
Sexual dysfunction	MPO, VMH
Decreased libido	MPO, VMH
Sex hormone dysregulation (for	MPO, VMH
example, leutinizing hormone,
follicle stimulating hormone
dysregulation)
Misregulated release of prolactin	PVN
Misregulated release of	SO, PVN
oxytocin/vasopressin
Misregulated release of leutinizing	MPO
hormone, follicle stimulating hormone
Misregulated release of somatostatin	Pe
Hypertension	AP, NTS, PVN, DMH,
	BST, CeA
Hypotension	AP, NTS, PVN, DMH,
	BST, CeA
Fluid imbalance	AP, NTS, PVN, DMH,
	BST, CeA

It is known in the art that currently available drugs for controlling absence seizures are effective in the prevention and treatment of a variety of neurologic and psychiatric conditions. For example, valproate, one of the most commonly used medications for controlling absence seizures, is also useful in the treatment of bipolar and schizoaffective disorders, depression, anxiety, alcohol withdrawal and dependence, agitation associated with dementia, impulsive aggression, neuropathic pain, and for the prophylactic treatment of migraine (see, for example, Loscher, Prog. Neurobiol. 58:31-59 (1999), and Davis et al., J. Clin. Psychopharmacol. 20:1S-17S (2000)). Thus, PrRP receptor agonists and antagonists can be used to treat conditions in which other anti-absence seizures drugs are effective.

The PrRP agonists and antagonists of the invention can be formulated and administered by those skilled in the art in a manner and in an amount appropriate for the condition to be treated; the weight, gender, age and health of the individual; the biochemical nature, bioactivity, bioavailability and side effects of the particular compound; and in a manner compatible with concurrent treatment regimens. An appropriate amount and formulation for controlling absence seizures in humans can be extrapolated based on the activity of the compound in the assays described herein. Similarly, an appropriate amount and formulation for promoting wakefulness or sleep in humans can be extrapolated based on the activity of the compound in the assays described herein. An appropriate amount and formulation for use in humans for other indications can be extrapolated from credible animal models known in the art of the particular disorder.

The total amount of compound can be administered as a single dose or by infusion over a relatively short period of time, or can be administered in multiple doses administered over a more prolonged period of time. Additionally, the compounds can be administered in slow-release matrices, which can be implanted for systemic delivery or at the site of the target tissue. Contemplated matrices useful for controlled release of therapeutic compounds are well known in the art, and include materials such as DepoFoam™, biopolymers, micropumps, and the like.

The compounds and compositions of the invention can be administered to the subject by any number of routes known in the art including, for example, intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisternally, intra-articularly, intracerebrally, orally, intravaginally, rectally, topically, intranasally, or transdermally. A preferred route for humans is oral administration.

A PrRP receptor agonist or antagonist can be administered to a subject as a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. Those skilled in the art understand that the choice of a pharmaceutically acceptable carrier depends on the route of administration of the compound and on its particular physical and chemical characteristics. Pharmaceutically acceptable carriers are well known in the art and include sterile aqueous solvents such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils such as olive oil and injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that stabilize the compound, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include carbohydrates such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins.

For applications that require the compounds and compositions to cross the blood-brain barrier, formulations that increase the lipophilicity of the compound are particularly desirable. For example, a PrRP receptor agonist or antagonist can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

A PrRP receptor agonist or antagonist can also be prepared as nanoparticles. Adsorbing peptide compounds onto the surface of nanoparticles has proven effective in delivering peptide drugs to the brain (see Kreuter et al., Brain Res. 674:171-174 (1995)). Exemplary nanoparticles are colloidal polymer particles of poly-butylcyanoacrylate with PrRP adsorbed onto the surface and then coated with polysorbate 80.

In current absence seizure treatment regimes, more than one compound is often administered to an individual for maximal seizure control. Thus, for use in controlling absence seizures, a PrRP receptor agonist can advantageously be formulated with a second compound that controls absence seizures. Such compounds include, for example, valproate, ethosuximade, flunarizine, trimethadione and lamotrigine. Contemplated methods of controlling absence seizures include administering the compounds and compositions of the invention alone, in combination with, or in sequence with, such other compounds.

Similarly, in current sleep disorder treatment regimes, more than one compound is often administered to an individual for maximal reduction in symptoms. Thus, for use in promoting wakefulness or sleep, a PrRP receptor agonist or antagonist can be formulated with a second compound that promotes wakefulness or sleep. Compounds that promote wakefulness include, for example, amphetamine, methylphenidate, ephedrine, cathinone and caffeine. Sleep attacks associated with narcolepsy generally respond to stimulants such as methylphenidate (Ritalin), modafinil, and amphetamines such as dextroamphetamine, mazindol and selegiline. Therefore, a PrRP receptor agonist can be combined with such compounds to effectively promote wakefulness in individuals having narcolepsy.,

Well known compounds that promote sleep include, for example, opiates, barbiturates, benzodiazepines, and anesthetics. A PrRP receptor antagonist that induces sleep can be combined with such compounds to effectively promote sleep, or can be used to increase the efficacy of sleep-promoting compounds, thereby reducing the required dosage of compounds that are addictive or have unwanted side-effects.

In addition, a PrRP receptor agonist or antagonist can be formulated with a compound that reduces a symptom associated with the treated disorder. For example, a combination of a PrRP receptor agonist can be combined with a compound effective for reducing cateplexy, such as a dopamine receptor D2/D3 antagonist, to provide a composition useful for treating narcolepsy. Contemplated methods of promoting wakefulness or sleep include administering the compounds and compositions of the invention and PrRP receptor agonists and antagonists alone, in combination with, or in sequence with, such other compounds.

Claim 1 of 4 Claims

1. A method of screening for a compound for promoting wakefulness in a mammal, comprising:

(a) contacting a PrRP receptor with one or more candidate compounds under conditions wherein PrRP promotes interaction of PrRP receptor with an AMPA receptor associated protein;

(b) identifying a compound that promotes said interaction;

(d) determining the ability of said compound to promote wakefulness.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]