WO2006012403A1

WO2006012403A1 - Methods of treatment: cell signaling and glutamate release

Info

Publication number: WO2006012403A1
Application number: PCT/US2005/025853
Authority: WO
Inventors: Mark F. Bear
Original assignee: Massachusetts Institute Of Technology
Priority date: 2004-07-20
Filing date: 2005-07-20
Publication date: 2006-02-02

Abstract

In one embodiment, subjects with mental retardation, Down's syndrome, fragile X syndrome and autism are treated with at least one compound that down regulates endocannabinoid signaling. In another embodiment, subjects are treated with at least one agonist selected from the group consisting of a Group II mGluR agonist and a Group III mG1uR agonist. In a further embodiment, a human is treated with a compound that down regulates presynaptic glutamate release.

Description

METHODS OF TREATMENT: CELL SIGNALING AND GLUTAMATE RELEASE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos: 60/589,671, filed July 20, 2004 and 60/678,518, filed May 6, 2005. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

In the nervous system, cell signaling includes the release of an intracellular signaling molecule, a neurotransmitter, such as glutamate, from a presynaptic neuron into the synaptic cleft. Once released, a neurotransmitter can mediate cellular signaling by affecting postsynaptic neurons. Certain conditions (e.g., mental retardation, Down's syndrome, autism and fragile X syndrome) in subjects, such as humans, have been associated with or believed to be a consequence of inappropriate neuronal cell signaling. Currently available treatments for certain conditions, such as mental retardation, fragile X syndrome, Down's syndrome and autism, may not result in appropriate neuronal cell signaling. Thus, there is a need to develop new, improved and effective methods of treatment of conditions associated with or believed to be a consequence of inappropriate cell signaling.

SUMMARY OF THE INVENTION

The present invention is directed to methods of treating subjects, including human subjects having mental retardation, fragile X syndrome, Down's syndrome and autism.

In one embodiment, the invention is a method of treating a subject, comprising the step of administering at least one compound that down regulates endocannabinoid signaling to a subject having at least one condition selected from the group consisting of mental retardation, Down's syndrome, fragile X syndrome and autism. In another embodiment, the invention is a method of treating a subject, comprising the step of administering at least one agonist selected from the group consisting of a Group II niGluR agonist and a Group III mGluR agonist.

In still another embodiment, the invention is a method of treating a human, comprising the step of administering a compound that down regulates presynaptic glutamate release to a subject.

The invention described herein is a method for treating subjects, including humans having mental retardation, fragile X syndrome, Down's syndrome and autism. Appropriate neuronal signaling can be restored to thereby halt, reverse or diminish the progression of inappropriate neuronal signaling to increase the quality of life for the subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts neuronal signaling between presynaptic and postsynaptic neurons in the endocannabinoid signaling pathway. Figure 2 depicts neuronal signaling of presynaptic neurons expressing Group

II and Group III mGluRs.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.

The present invention is directed to methods of treating subjects, including humans with mental retardation, fragile X syndrome, Down's syndrome and autism. The subject having fragile X syndrome can also have autism.

As previously reported, Group I mGluR (metabotropic glutamate receptor) agonists increase long term depression (LTD) in brains obtained from fragile X mental retardation protein (FMRP) knockout (KO) mice (Huber, K.M., et al., Proc. Natl. Acad. Sci.USA). It is believed that increases in LTD may be a consequence of elevated glutamate release that activates Group I mGluR signaling. Elevated or irregular LTD (LTD not within a normal range) may be associated with inappropriate neural signaling in certain conditions, such as mental retardation, fragile X syndrome, Down's syndrome and autism. Thus, it is believed that normalization of LTD of Group I mGluR can attenuate conditions in subjects. Normalization of LTD can be through cell signaling pathways that decrease synaptic glutamate, including down regulation of endocannabinoid signaling and activation of Group π mGluR and Group III mGluR. mGluRs are a heterogeneous family of glutamate G-protein coupled receptors. mGluRs are classified into three groups. Group I receptors (mGluRl and mGluRS) can be coupled to stimulation of phospholipase C resulting in phosphoinositide hydrolysis and elevation of intracellular calcium levels, modulation of ion channels (e.g.,potassium channels, calcium channels, non-selective cation channels) and N-methyl-D-aspartate (NMDA) receptors. mGluR5 can be present on a postsynaptic neuron. niGlurl can be present on a presynaptic neuron and/or a postsynaptic neuron. Group II receptors (niGluR2 and niGluR3) and Group III receptors (mGluRs 4, 6, 7, and 8) inhibit cAMP formation and G-protein-activated inward rectifying potassium channels. Group II mGluRs and Group III mGluRs are negatively coupled to adenylyl cyclase, generally present on presynaptic neurons, but can be present on postsynaptic neurons and function as presynaptic autoreceptors to reduce glutamate release from presynaptic neurons.

In one embodiment, the invention is a method of treating a subject, comprising the step of administering at least one compound that down regulates endocannabinoid signaling to a subject having at least one condition selected from the group consisting of mental retardation, Down's syndrome, fragile X syndrome and autism.

"Down regulates," as used herein, refers to any decrease or any inhibition in a cellular process or a cellular event or intermediate in a cellular event associated with the biological or cellular process (e.g., endocannabinoid signaling, protein synthesis, glutamate release). In one embodiment, compounds that down regulate -A-

endocannabinoid signaling are employed in the methods of the invention. In another embodiment, compounds that down regulate presynaptic glutamate release are employed in the methods of the invention. Compounds that down regulate endocannabinoid signaling can down regulate presynaptic glutamate release. Down regulation of endocannabinoid signaling can be, for example, the prevention or any decrease in binding of a signal external to a cell (a first messenger), such as a ligand (e.g., glutamate), to a endocannabinoid receptor (e.g., a presynaptic CBl receptor). Down regulation can be disruption of a cellular process following binding of an external signal (e.g., ligand) to an endocannabinoid receptor, such as the prevention of activation of adenylyl cyclase, phospholipase C (PLC) or phospholipase D (PLD). Down regulation can also be disruption of a cellular process following binding of an external signal to an endocannabinoid receptor, such as the prevention of activation of a G-protein (Gs, Gq), a decrease in a G-protein (Gs, Gq) activation, prevention of activation of second messengers activated by endocannabinoid receptors (e.g., cAMP, IP₃, diacylglycerol (DAG), PLC, PLD) or a decrease in the activity of an intracellular effector, such as a cAMP -dependent protein kinase, protein kinase C (PKC) or calcium release. Figure 1 depicts down regulation of endocannabinoid signaling by, for example, the use of inhibitors (also referred to herein as "antagonists") of presynaptic CBl receptors and thereby treatment of mental retardation, fragile X syndrome, Down's syndrome and autism in a human.

Compounds used in the methods of the invention that down regulate endocannabinoid signaling can interfere with binding of a ligand (e.g., endocannabinoid) to an endocannabinoid receptor (e.g., CBl receptor). "Interfere," as used herein when referring to binding of a ligand to a receptor, means any effect on ligand-receptor binding that occurs in the presence of the compound that prevents the ligand from activating the receptor or mediating cell signaling.

Compounds used in the methods of the invention that down regulate endocannabinoid signaling can alter processes in cell signaling pathways, such as cAMP-dependent protein kinase. "Alter," as used herein in reference to cell signaling pathways, means any difference in the cell signaling pathway, molecule or intermediate in the cell signaling pathway that occurs in the presence of the compound that is not observed in the absence of the compound. An alteration can be, for example, a structural change in a protein kinase that inhibits activity of a protein kinase that was activated by ligand binding to a cell surface receptor, such as an endocannabinoid receptor or a Group II mGluR or a Group III mGluR. Compounds that down regulate endocannabinoid signaling can be antagonists of endocannabinoid receptors, such as a CBl endocannabinoid receptor antagonist. The CBl endocannabinoid receptor antagonists can be natural or synthetic antagonists of CBl receptors. Suitable CBl endocannabinoid antagonists are, for example,

N-(Piperidin-l-yl)-5-(4-iodophenyl)-l-(2₅4-dichlorophenyl)-4-methyl-lH-pyrazole-3 -carboxamide (AM251),

1 -(2,4-Dichlorophenyl)-5 -(4-iodophenyl)-4-methyl-N-4-morpholinyl- 1 H-pyrazole-3- carboxamide (AM281), and 5-(4-Chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-l-yl)-lH-pyrazole -3-carboxamide (SR141716A).

An antagonist of an endocannabinoid receptor is a substance that diminishes or abolishes the effect of a ligand (an endocannabinoid or agonist of an endocannabinoid) that activates an endocannabinoid receptor. The endocannabinoid antagonist may act at the level of ligand-receptor interaction, such as by competitively or non-competitively (e.g., allosterically) inhibiting endocannabinoid binding. The endocannabinoid antagonist can be, for example, a chemical antagonist or a pharmacokinetic antagonist. The endocannabinoid antagonist may act downstream of the receptor, such as by inhibiting receptor interaction with a G- protein or subsequent cell signaling events associated with G-protein activation, such as activation of PLC, an increase in intracellular calcium, the production of or levels of cAMP or adenyl cyclase and stimulation or modulation of ion channels (e.g., potassium channels, calcium channels).

A pharmacokinetic antagonist can decrease the concentration of a ligand (e.g., endocannabinoid) at a site of action, for example, by increasing the rate of metabolic degradation of the active ligand. Antagonism by a compound that reduces endocannabinoid signaling can involve reversible competitive antagonism or irreversible, or non-equilibrium, competitive antagonism. Reversible competitive antagonism occurs when the rate of dissociation of the antagonist from the receptor is sufficiently high that, on addition of the ligand, the antagonist molecules binding the receptors are effectively replaced by the ligand. Irreversible or non-equilibrium competitive antagonism occurs when the antagonist dissociates very slowly or not at all from the receptor, with the result that no change in the antagonist occupancy takes place when the ligand is applied. A competitive antagonist refers to a compound that binds to the endocannabinoid receptor or to the endocannabinoid signaling ligand in a manner that sterically interferes with the interaction of the endocannabinoid with the receptor.

In non-competitive antagonism, the endocannabinoid antagonist does not compete directly with endocannabinoid binding at the receptor, yet blocks a point in the signal transduction pathway subsequent to receptor activation by the ligand. Down regulation of endocannabinoid signaling can also be through activation of Group II and/or Group III mGluRs by, for example, agonists of presynaptic Group II and Group III mGluRs (Figure 2). An "agonist," as used herein, is a compound that activates cell signaling. The agonist (also referred to herein as an activator) can be a natural or synthetic ligand for a Group II mGluR or Group III mGluR. Activation of cell signaling can be, for example, an increase in the affinity or binding of a signal external to a cell (a second messenger), such as a ligand (e.g., glutamate) for a Group II and/or a Group III mGluR (e.g., a presynaptic Group II and/or Group III mGluR). Activation of cell signaling can be any increase or augmentation of a cellular process following binding of an external signal (e.g., a ligand) to a Group II and/or Group III mGluR, such as activation of adenyl cyclase, PLC or PLD. Activation of cell signaling can also be any increase in a cellular process following binding of an external ligand to a Group II and/or Group III mGluR, such as activation of a G-protein, activation of second messengers (e.g., cAMP, IP₃, DAG, PLC, PLD) or any increase in the activity or amount of an intracellular effector, such as a cAMP-dependent protein kinase, PKC or calcium release. Figure 2 depicts activation of Group II mGluR and/or Group III mGluR by, for example, a Group II niGluR agonist and/or a Group III mGluR agonists and thereby treatment of a subject.

Examples of Group I and Group II mGluR agonists for use in the invention include LY354740, L-AP4 (Capogna, M. Eur. J. Neurosci. 19: 2847-2858 (2004), the teachings of which are hereby incorporated by reference in their entirety)

(2R,4R)-4-Aminoρyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC),

(2S,rS,2'S)-2-(Carboxycycloρropyl)glycine (L-CCG-I)₅

N-Acetyl-L-aspartyl-L-glutamic acid (Spaglumic acid),

(S)-3-Carboxy-4-hydroxyρhenylglycine ((S)-3C4HPG), (S)-4-Carboxy-3-hydroxyphenylglycine ((S)-4C3HPG).

In another embodiment, the invention is a method of treating a subject, comprising the step of administering at least one agonist selected from the group consisting of a Group II mGluR agonist and a Group III mGluR agonist. The Group π mGluR agonist and Group III mGluR agonist can be an agonist of a presynaptic neuron and/or a postsynaptic neuron of a Group II mGluR and/or a Gropu III mGluR.

In a further embodiment, the invention is a method of treating a human comprising the step of administering a compound that down regulates presynaptic glutamate release to the human. The compound that down regulates presynaptic glutamate release can be an endocannabinoid antagonist (e.g., a presynaptic CBl receptor agonist), a Group II mGluR antagonist and/or a Group III mGluR antagonist.

A subject treated by the methods described herein can be any mammal, such as a rodent (e.g., rat, mouse, guinea pig) or a primate (e.g., monkey, baboon, human). In one embodiment, the methods of the invention can be employed to treat humans with mental retardation. Mental retardation is characterized by a significantly below-average score on a test of mental ability or intelligence and by limitations in the ability to function in areas of daily life, such as communication, self-care, and getting along in social situations and school activities. Mental retardation can be caused, for example, by injury, disease or a brain abnormality that can occur before a child is born, during childhood or during adulthood. Some of the most common known causes of mental retardation are genetic causes, as in Down's syndrome and fragile X syndrome. Other causes that take place before a child is born include genetic conditions, fetal alcohol syndrome, infections or birth defects that affect the brain. In another embodiment, the methods of the invention can also be employed to treat fragile X syndrome, a genetic form of mental retardation. Fragile X syndrome is diagnosed by a genetic test that determines the number of CGG repeats in the 5' untranslated region of the fragile X mental retardation (FMRl) gene that results in transcriptional silencing of the fragile X mental retardation protein

In another embodiment, the methods of the invention can be employed to treat Down's Syndrome, another genetic cause of mental retardation. Humans with Down's syndrome have a tri-semi-chromosome 21 failure of chromosomes to separate. In yet another embodiment, the methods of the invention can be employed to treat autism. Characteristics of autism include deficits in social skills, and verbal and non-verbal communication and can be accompanied by restricted, repetitive or stereotypic behavior.

A human with autism can also have fragile X syndrome. One of skill in the art would be capable of diagnosing a subject with mental retardation, fragile X syndrome, Down's syndrome and autism who would benefit from the methods of the invention.

An "effective amount," also referred to herein as a "therapeutically effective amount," when referring to the amount of a compound or composition (e.g., pharmaceutical composition containing a compound) for use in the methods of the invention, is defined as that amount, or dose, of a compound or composition that, when administered to a subject, is sufficient for therapeutic efficacy (e.g., an amount sufficient decrease glutamate release, to exhibit a clinical improvement in a behavior or mental cognitive test score). The methods of the present invention can be accomplished, for example, by the administration of a compound by enteral or parenteral means. Specifically, the route of administration is by oral ingestion (e.g., tablet, capsule form). Other routes of administration as also encompassed by the present invention including intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous routes and nasal administration. Suppositories or transdermal patches can also be employed. One or more compounds that down regulate endocannabinoid, activate

Group II niGluR (e.g., a ligand or agonist of Group II mGluR), activate Group III niGluR (e.g., a ligand or agonist of Group III mGluR) or decrease presynaptic glutamate can be administered to the subject. For example, the subject can be treated with a compound that down regulates endocannabinoid signaling by preventing binding of endocannabinoid to a presynaptic CB 1 receptor or a compound that disrupts intracellular signaling following binding of an endocannabinoid to an endocannabinoid receptor. Likewise, a compound that decreases presynaptic glutamate can increase binding of a ligand or an agonist of a Group π mGluR or a ligand or an agonist of a Group III mGluR to a presynaptic neuron.

Compounds that down regulate endocannabinoid signaling, activate Group II mGluR, activate Group III mGluR or decrease presynaptic glutamate can be co-administered. Coadminstration can include simultaneous or sequential administration of such compounds. Compounds that down regulate endocannabinoid signaling, activate Group II mGluR, activate Group III niGluR or decrease presynaptic glutamate can be administered alone or can be coadministered to the subject with another treatment regimen.

Compounds that down regulate endocannabinoid signaling, activate Group II mGluR, activate Group III mGluR or decrease presynaptic glutamate can be administered alone or as admixtures with conventional excipients, for example, pharmaceutically, or physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the compounds. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the compound used in the methods of the invention. The preparations can also be combined, when desired, with other active substances to reduce metabolic degradation. The compound for use in the methods of the invention can be administered in a single or in more than one dose over a period of time to confer the desired effect (e.g., alleviate urges and repetitive actions). The compounds can be administered in a dose of between about 0.5 mg/kg to about 5 mg/kg body weight; or between about 1 mg/kg to about 10 mg/kg body weight. The compounds can be administered in doses of about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 700 mg, or about 900 mg. The compounds can be administered once a day or multiple times per day.

When parenteral application is needed or desired, particularly suitable admixtures for a compound that down regulates endocannabinoid, activate Group II mGluR, activate Group III niGluR or decrease presynaptic glutamate are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. In particular, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and the like. Ampules are convenient unit dosages. The compound used in the methods of the invention can also be incorporated into liposomes or administered by transdermal pumps or patches. Pharmaceutical admixtures suitable for use in the present invention are well-known to those of skill in the art and are described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309 the teachings of which are hereby incorporated by reference.

The dosage and frequency (single or multiple doses) administered to a subject can vary depending upon a variety of factors, including, for example, the condition being treated, duration of the condition being treated, the duration of OCD and obsessions and compulsions associated with OCD, age, sex, health, body weight and body mass index of the subject; kind of concurrent treatment, or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods of the present invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.

EQUIVALENTS

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a subject, comprising the step of administering at least one compound that down regulates endocannabinoid signaling to a subject having at least one condition selected from the group consisting of mental retardation, Down's syndrome, fragile X syndrome and autism.

2. The method of Claim 1 , wherein the subject is a human.

3. The method of Claim 1 , wherein the compound that down regulates endocannabinoid signaling is an endocannabinoid antagonist.

4. The method of Claim 2, wherein the human has fragile X syndrome.

5. The method of Claim 2, wherein the human has autism.

6. The method of Claim 2, wherein the human has Down's syndrome.

7. The method of Claim 1 , wherein the compound that down regulates endocannabinoid signaling is a Group II mGluR agonist.

8. The method of Claim 1 , wherein the compound that down regulates endocannabinoid signaling is a Group III mGluR agonist.

9. A method of treating a subject, comprising the step of administering at least one agonist selected from the group consisting of a Group II mGluR agonist and a Group III mGluR agonist.

10. The method of Claim 9, wherein the subject is a human.

11. The metiiod of Claim 10, wherein the human has at least one condition selected from the group consisting of mental retardation, Down's syndrome, fragile X syndrome and autism.

12 . The method of Claim 9 wherein the agonist is a Group II mGluR agonist.

13. The method of Claim 9, wherein the agonist is a Group HI mGluR agonist.

14. A method of treating a human, comprising the step of administering a compound that down regulates presynaptic glutamate release to the human.

15. The method of Claim 14, wherein the human has at least one condition selected from the group consisting of mental retardation, Down's syndrome, fragile X syndrome and autism.

16. The method of Claim 14, wherein the compound is an endocannabinoid antagonist.

17. The method of Claim 14, wherein the compound is a Group II mGluR agonist.

18. The method of Claim 14, wherein the compound is a Group III mGluR agonist.