This invention provides in vivo and in vitro methods of screening for an agent or a combination of agents that reduces one or more repetitive behaviors, comprising contacting neuronal cells of an animal with a HOXB8 gene mutation with the agent combination of agents to be screened, and determining whether one or more repetitive behaviors of the animal is reduced or whether one or more biochemical correlates of repetitive behaviors is reduced, the reduction in one or more repetitive behaviors or biochemical correlates indicating an agent or combination of agents that reduces repetitive behaviors. The invention also provides method of treating a subject with repetitive behaviors, comprising administering a therapeutically effective dose of the agent or combination of agents identified by the screening method. Further provided is a population of animals with a HOXB8 gene mutation, wherein more than 30% of the animals have excessive grooming behaviors and wherein less than 10% of the animals show skeletal defects.

Description of the Invention

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to an in vivo method of screening for an agent or combination of agents that reduces one or more repetitive behaviors, comprising contacting neuronal cells of an animal with a HOXB8 gene mutation with the agent or combination of agents to be screened, and determining whether one or more repetitive behaviors of the animal is reduced, the reduction in one or more repetitive behaviors indicating an agent or combination of agents that reduces repetitive behaviors. The invention also relates to an in vitro method of screening for an agent or combination of agents that reduces one or more repetitive behaviors, comprising contacting neuronal cells of an animal with a HOXB8 gene mutation with the agent or combination of agents to be screened, and determining whether one or more biochemical correlates of repetitive behaviors of an animal is reduced, the reduction in one or more repetitive behaviors indicating an agent or combination of agents that reduces repetitive behaviors.

The invention also relates to a method of treating a subject with repetitive behaviors, comprising administering a therapeutically effective dose of the agent or combination of agents identified by the screening method.

In another aspect, the invention relates to a population of animals with a HOXB8 gene mutation, wherein more than 30% of the animals have excessive grooming behaviors and wherein less than 10% of the animals show skeletal defects.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes combinations or mixtures of various agents, and the like.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

By the term "therapeutically effective amount" of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired regulation of the gene expression or regulation of a particular repetitive behavior. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

"Eliciting," "modulating," "regulating," selective gene expression is intended to mean that a compound is capable of acting as an activator or an antagonist of gene expression, either directly or indirectly. For example, gene expression could be regulated at the level of DNA transcription or translation.

In one embodiment, the present invention provides a method of screening for an agent or combination of agents that reduces one or more repetitive behaviors. The screening method comprises the steps of contacting neuronal cells of an animal with a HOXB8 gene mutation with the agent or combination of agents to be screened, and determining whether one or more repetitive behaviors of the animal is reduced. The reduction in one or more repetitive behaviors indicates an agent or combination of agents that reduces repetitive behaviors.

The agent or combination of agents used in the screening method may be used in a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, Pa., which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that may be used in conjunction with the preparation of formulations of the agents and which is incorporated by reference herein.

By "repetitive behaviors" is meant any number of repetitive compulsive-like behaviors, including excessive grooming behaviors. Such repetitive actions in human have been described in Diagnostic and Statistical Manual of Mental Disorders, ed 4, 1994 (DSM-IV)) and include excessive hand washing, excessive bathing and other ritualistic behaviors, some of which are aimed at cleanliness (DSM-IV). Repetitive actions in animals are similar and include mutilatory biting and picking of their skin, as well as pulling and plucking of their hair. Such repetitive behaviors can be self directed or can be directed toward others. By "excessive grooming behaviors" is meant a significantly higher incidences or duration of grooming behaviors as compared to control. The significantly higher amount can include an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater, or any amount in between.

Thus, in various embodiments of the screening method, the reduction in one or more repetitive behaviors comprises a reduction in one or more excessive grooming behaviors. For example, in one embodiment, the excessive grooming behavior comprises a reduction in the total amount of time the animal grooms. In another embodiment, the reduction in excessive grooming behavior comprises a reduction in the total number of times the animal initiates grooming. In another embodiment, the reduction in excessive grooming behavior comprises a reduction in hair removal caused by grooming. In yet another embodiment, the reduction in excessive grooming behavior comprises a reduction in lesions caused by grooming. The reduction in excessive grooming behavior comprises either a reduction in self-grooming, a reduction in grooming of other animals, or both.

The reduction in one or more repetitive behaviors can be determined in a variety of ways. For example, the animal to be tested could be observed or videotaped before and after the agent is administered, or the animal to be tested could be observed or videotaped after the agent is administered and compared to untreated animals with the same mutation. The number of repetitive behaviors before and after administration could be compared or the total amount of time engaged in the repetitive behavior could be determined.

The excessive grooming behaviors demonstrated in the animal to be screened can be non-induced or induced. By "induced" is meant that a causative agent that provokes grooming is used. Causative agents include, for example, stress or application of a stimulus to the animal's skin or coat. For example, a water mist applied to a rodent induces grooming.

By "contacting" is meant an instance of exposure of at least one neuron or neuronal cell to an agent. As used herein, the contacting step can be in vivo, e.g., where behavioral assessments of repetitive behaviors are used, or in vitro, e.g., when changes at the cellular level are assessed In vitro screening methods could include isolation and molecular characterization of individual neuronal phenotypes and screening of these specific cells types for biochemical changes that correlate with reduced repetitive behaviors in the whole animal. The screening assay could include high throughput analysis using microarrays and proteonomics.

In in vitro screening methods, the cell can be contacted with an agent, for example, by adding the agent to the culture medium (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the agent). In in vivo screening methods, the cell is contacted by adding the agent to the extracellular fluid of the neuronal cell (by orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, topically, transdermally, locally, systemically, intraventricularly, intracerebrally, subdurally, or intrathecally administering the agent to be screened). Depending upon the agent to be screened and the mode of administration, one skilled in the art would recognize that the blood brain barrier may diminish or prevent access to neurons when administered systemically. One skilled in the art would know to modify the mode of administration, the pharmacologic carrier, or other parameters to circumvent restrictions posed by the blood brain barrier.

One method of in vivo administration is the use of micro-osmotic pumps (e.g., Alzet.RTM. pump). The pumps can be placed under the animals skin with a cannula extending into the subdural or intraventricular space.

The duration of "contact" with a cell or group of cells is determined by the time the agent is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell or cells. Such duration of contact varies based on the half-life of the agent. The contacting step in vivo can be by bolus delivery or by continuous infusion.

The neuronal cell contacted in the screening method may or may not be a Hoxb8 expressing cell. Given the structure of neuronal circuitry, the pharmacologic effect of an agent may be upstream or downstream of a Hoxb8 expressing cell. For example, an agent that reduces repetitive behaviors could affect a serotonergic neuron which synapases with a Hoxb8 expressing cell.

By "a HOXB 8 gene mutation" is meant any mutation in the HOXB8 gene that results in a decrease in gene function. A decrease in gene function includes a decrease in transcription or expression. Such a mutation includes a nonsense mutation or a mutation that results in a hypomorphic allele.

The animal with the HOXB8 gene mutation is preferably a transgenic mouse, but can be any laboratory animal in which similar gene manipulation can be accomplished using techniques well known in the art. Preferably the mutation is achieved by point mutation rather than by the introduction of exogenous DNA, for example, using the lacZ fusion method.

The invention also provides a population of animals with a HOXB8 gene mutation, wherein more than 30% of the animals have excessive grooming behaviors and wherein less than 10% of the animals show skeletal defects. The skeletal defects include patterning defects in the axial skeleton, like rib mutations (e.g., partial to fall fusion of two or more ribs, absence of all or part of one or more ribs, bifurcation of ribs). In the preferred embodiment, the HOXB8 gene mutation is a point mutation produced by gene targeting and is not a mutation that introduces exogenous DNA.

In another embodiment, more than 50% of the population of animals with the HOXB8 gene mutation show excessive grooming behaviors. In another embodiment, 75% of the animals show excessive grooming behaviors. In yet another embodiment, 100% of the animals show excessive grooming behaviors. The excessive grooming behaviors of the animals of the population comprise an increased total amount of time the mutant animal grooms as compared to animals lacking the mutation, an increased total number of times the mutant animal initiates grooming as compared to animals lacking the mutation, an increased hair removal caused by the mutant animal's grooming as compared to animals lacking the mutation, increased lesions caused by the mutant animal's grooming as compared to animals lacking the mutation, increased self-grooming, and/or increased mutant animal grooming of other animals. The excessive grooming behavior of the population of animals can be non-induced or induced.

The invention also provides a method of treating a subject with repetitive behaviors, comprising administering to the subject a therapeutically effective dose of the agent or combination of agents identified by the screening method of the invention. The repetitive behaviors being treated include, for example, compulsive behaviors. The repetitive behaviors of the subject may be symptoms of an obsessive compulsive disorder, Tourette's syndrome, trichotillomania, autism, disorders of mental delay (e.g., mental retardation) or other disorders or syndromes marked by repetitive actions such as those described in Diagnostic and Statistical Manual of Mental Disorders, ed 4, 1994 (DSM-IV).

The agents identified by the screening method of the invention may be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. The compounds may be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, topically, transdermally, locally, systemically, intraventricularly, intracerebrally, subdurally, or intrathecally. Depending upon the agent to be screened and the mode of administration, special provisions may be required to promote the agent to cross the blood brain barrier. One skilled in the art would know to modify the mode of administration, the pharmacologic carrier, or other parameters to circumvent restrictions posed by the blood brain barrier. The amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician.

Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example see Remington's Pharmaceutical Sciences, referenced above.

For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

Parental administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parental administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.

For topical administration, liquids, suspension, lotions, creams, gels or the like may be used as long as the active compound can be delivered to the surface of the skin.

The present invention also provides a method of screening for a genetic mutation associated with repetitive behaviors in a subject comprising acquiring from the subject a genetic sample and comparing that genetic sample with a genetic profile associated with a manifestation of repetitive behaviors. In an preferred embodiment, the genetic profile reflects a nonsense mutation or hypomorphic allele of the HOXB8 gene.
 

Claim 1 of 16 Claims

1. A method of screening for an agent or combination of agents that reduces one or more repetitive behaviors, comprising A. administering the agent or combination of agents to be screened to a mouse with a HOXB8 gene mutation, wherein the gene mutation is a frameshift mutation resulting in a premature stop codon within the first exon, or is an insertion of a floxed pMC-1neo.sup.r cassette into a homeodomain, and B. determining whether one or more repetitive behaviors of the mouse is reduced, the reduction in one or more repetitive behaviors indicating an agent or combination of agents that reduces repetitive behaviors.

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