Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/46—8-Azabicyclo [3.2.1] octane; Derivatives thereof, e.g. atropine, cocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/16—Aptamers

Definitions

• the present invention relates to methods for treating and/or preventing anxiety in a subject that involve administering small organic molecules or RNA aptamers.
• Anxiety can be described as a subjective feeling of unease, discomfort, apprehension or fearful concern accompanied by a host of autonomic and somatic manifestations. Anxiety can be a normal response to danger, but if it is prolonged, disproportionate in scale or duration, and interferes with normal daily activities, then a medical diagnosis of Anxiety Disorder is made. Extensive literature draws similarities between anxiety and fear, and also provides clear distinctions between these two different but overlapping phenomena. For example, fear in the absence of threat is anxiety.
• Anxiety disorder can be considered to occur when anxiety causes physiological symptoms.
• Tranquilizers are used to treat anxiety and work by slowing central nervous system activity. Their relaxing and calming effects have made them the most widely prescribed medication for anxiety. Antidepressants were developed to treat depression but are also effective for anxiety. Although these options begin to alter brain chemistry after the very first dose, their full effect requires a series of changes to occur. It can take about 4 to 6 weeks before symptoms start to fade. Therefore, it is necessary to take these medications continuously and long enough to let them work.
• High-potency benzodiazepines combat anxiety and have few side effects, other than drowsiness. Because people can become desensitized to benzodiazepines and may need increasing doses to get the same effect, the drugs are generally prescribed for short periods, especially for people who have abused drugs or alcohol and who become dependent on medication easily. An exception is people with panic disorder, who can take benzodiazepines for up to a year without harm.
• Anti-anxiety drugs like benzodiazepines work by reducing brain activity. While this temporarily relieves anxiety, it can also lead to unwanted side effects. Some people feel sleepy, foggy, and uncoordinated even on low doses of benzodiazepines. Other side-effects could include slurred speech, confusion, lightheadness, memory loss, forgetfulness, nausea and blurred vision.
• anti-anxiety tranquilizers include antidepressants, buspirone, and beta blockers.
• SSRIs selective serotonin reuptake inhibitors
• OCD obsessive-compulsive disorder
• depression depression
• SSRIs have fewer side effects than older antidepressants, but they sometimes produce slight nausea or agitation when people first start to take them.
• Buspirone (Buspar®), an azapirone, is a newer anti-anxiety tranquilizer used to treat General Anxiety Disorder. Possible side effects include drowsiness, dizziness, headaches, constipation, diarrhea, and nausea. Buspirone must be taken consistently for at least 2 weeks to achieve an anti-anxiety effect.
• Tricyclics are older than SSRIs. They are also started at low doses that are gradually increased. They sometimes cause dizziness, drowsiness, dry mouth, and weight gain, which can usually be corrected by changing the dosage or switching to another tricyclic medication.
• Monamine oxidase inhibitors are the oldest class of
• MAOIs antidepressant medications.
• Beta-blockers are often used to treat high blood pressure and heart conditions, but can also prevent the physical symptoms that accompany certain anxiety disorders. When a situation that can produce anxiety can be predicted (such as giving a speech), a doctor may prescribe a beta-blocker to keep physical symptoms of anxiety under control. Beta-blockers carry the potential side effects of light-headedness, sleepiness, nausea, and unusually slow pulse.
• a first aspect of the present invention is directed to a method that improves, prevents, or treats anxiety and anxiety symptoms in a subject
• This method involves selecting a subject in need of improvement, treatment, and/or prevention of anxiety and/or anxiety symptons, and administering to the selected subject a ligand that binds to a regulatory site on the extracellular domain of the nicotinic acetylcholine receptor and improves, prevents, or treats anxiety and anxiety symptoms in the subject.
• the present invention is directed to ligands, including small organic molecules and RNA aptamers of various types, that bind to specific sites on the nicotinic acetylcholine receptors. These ligands either inhibit (called Class 1 ligands) or alleviate (called Class 2 ligands) the inhibition of receptor channels and so inhibit or restore (despite the continued presence of the inhibitor) the function of the receptor in its role to promote nerve activity and health.
• ligands including small organic molecules and RNA aptamers of various types, that bind to specific sites on the nicotinic acetylcholine receptors. These ligands either inhibit (called Class 1 ligands) or alleviate (called Class 2 ligands) the inhibition of receptor channels and so inhibit or restore (despite the continued presence of the inhibitor) the function of the receptor in its role to promote nerve activity and health.
• Figure 1 is a graph showing brain (ng/g) and plasma (ng/ml) concentrations of ecgonine methyl ester ("EME”) in rats following intraperitoneal administration of a 10 mg/kg dose at 0, 1, 4, 8, and 24 hours.
• EME ecgonine methyl ester
• Figures 2A-2C are Morris water maze traces of three individual rats.
• Figure 2A shows the trace of a young control rat swimming in the Morris water maze bath for approximately 30 seconds searching for the platform, with no drug treatment and before training.
• Figure 2B shows the trace of an aged anxious rat swimming almost entirely at the perimeter of the water bath with no drug treatment and before training.
• Figure 2C shows the trace of the aged anxious rat of Figure 2B after 10 days of treatment with EME at lOmg/kg per day. All doses of drug were administered by intraperitoneal injection.
• Figure 3 is a graph measuring thigmotaxis, an index of anxiety in young rats (control), aged anxious rats, and aged anxious rats following treatment with 3mg/kg or 10 mg/kg of EME treatment.
• the diagram shows percent of time (y-axis) spent at the perimeter of the water bath ("hugging the walls"), with a high percentage indicating severe anxiety.
• the drug doses reduced the percent of time at the perimeter and therefore the severity of anxiety.
• Figure 4 is a graph showing a decline in the level of thigmotaxis as a function of time in the aged no-treatment control animals, the aged animals treated with 3 mg/kg of EME, and the aged animals treated with 10 mg/kg of EME as described in Figure 3.
• a first aspect of the present invention is directed to a method that improves, prevents, or treats anxiety and anxiety symptoms in a subject.
• This method involves selecting a subject in need of improvement, treatment, and/or prevention of anxiety and/or anxiety symptoms, and administering to the selected subject a ligand that binds to a regulatory site on a nicotinic acetylcholine receptor and improves, prevents, or treats anxiety and anxiety symptoms in the subject.
• a subject suitable for treatment using methods of the present invention includes any animal, preferably a mammal, e.g., human, non-human primate, rodent, cow, horse, sheep, pig, goat, deer, elk, bison, etc.
• Anxiety as used herein includes any state or condition that causes anxiety behavior or concern by a subject.
• the intent of this invention is to treat or modify any anxiety behavior and symptoms caused by conditions that include, but are not limited to, the following anxiety-related disorder types in a subject: dissociative, eating, impulse- control, adjustment, anxiety, mood, personality, psychotic, post-traumatic stress, sexual, sleep-related, and somatoform disorders.
• DSM V Statistical Manual of Mental Disorders
• Anxiety can be described as a subjective feeling of unease, discomfort, apprehension or fearful concern accompanied by a host of autonomic and somatic manifestations. Anxiety can be a normal response to danger, but if it is prolonged, disproportionate in scale or duration, and interferes with normal daily activities, then a medical diagnosis of Anxiety Disorder is made. Extensive literature draws similarities between anxiety and fear, and also provides clear distinctions between these two different but overlapping phenomena. For example, fear in the absence of threat is anxiety. Anxiety disorder can be considered to occur when anxiety causes physiological symptoms.
• the patient also experiences a wide variety of somatic phenomena, such as palpitations, sweating, headache, tachypnea, trembling, chest pain, urinary frequency, motor tension, trouble swallowing, sleep problems, irritability, nausea and dizziness. Emotional and physical sensations together can lead to problems in cognition (learning, memory, concentration, decision-making ability etc.).
• anxiety disorders commonly includes sub-classifications of panic, phobia, post-traumatic symptom disorder, acute stress, substance-abuse linked anxiety, and many others.
• anxiety can be improved and anxiety in a subject can be treated or prevented by administering to the subject a ligand that binds to the nicotinic acetylcholine receptor and treats or prevents anxiety or anxiety symptoms in a subject.
• Laboratory work has demonstrated the existence of a regulatory site within the extracellular domain of the nicotinic
• ligand includes, but is not limited to, small organic molecules, aptamers, and other compounds that similarly bind to this regulatory site on the nicotinic acetylcholine receptor and induce an allosteric change in the receptor, thereby improving or enhancing the flow of inorganic cations through the ion channel of the receptor.
• the enhanced flow of inorganic cations through the receptor channel improves nerve function and relieves or prevents anxiety.
• Noncompetitive Inhibitors (MK-801 and Cocaine) on a Protein (Nicotinic Acetylcholine Receptor)-Mediated Reaction," Biochemistry 42:6106-61 14 (2003), Cui et al, "Selection of 2'-Fluoro-modified RNA Aptamers for Alleviation of Cocaine and MK-801 Inhibition of the Nicotinic Acetylcholine Receptor," J. Membrane Biol.
• Ligands that bind to the regulatory site of the nicotinic acetylcholine receptor comprise two different classes.
• Class 1 ligands are compounds that bind with higher affinity to the regulatory site on the closed-channel form than on the open-channel form of the receptor. Class 1 ligands facilitate closure and/or continued existing closure of the receptor ion channel, which inhibits neurotransmission and impairs cognition. Class 1 ligands include both endogenous and exogenous compounds. Prototypical exogenous Class 1 ligands include, without limitation, cocaine, MK-801, and phencyclidine.
• Class 2 ligands are compounds that bind to the regulatory site on the nicotinic acetylcholine receptor and shift the channel-opening equilibrium towards the open channel form of the receptor. For example, in the presence of an activating ligand such as acetylcholine or carbamoylcholine, and in the presence of a deleterious factor such as a Class 1 ligand, a mutation, etc. Class 2 ligands bind with equal or higher affinity to the regulatory site on the open-channel form of the receptor than to the closed- channel form. This binding shifts the channel-opening equilibrium to the open-channel state and alleviates the inhibition and impairment caused by a Class 1 compound, mutation, etc.
• an activating ligand such as acetylcholine or carbamoylcholine
• Class 2 ligands bind with equal or higher affinity to the regulatory site on the open-channel form of the receptor than to the closed- channel form. This binding shifts the channel-opening equilibrium to the open-channel state and
• Exemplary Class 2 ligands suitable for use in accordance with the methods of the present invention include, without limitation, tropane and its derivative, e.g. , ecgonine, ecgonine methyl ester, RTI-4229-70, RCS-III-143, RCS-III- 140A, RCS-III- 218, and RCS-III-202A, piperidine and its derivatives, derivatives of MK801 (but not MK-801), derivatives of phencyclidine (but not phencyclidine), and certain RNA aptamers all of which are described in more detail infra.
• These Class 2 ligands are the anti-anxiety ligands that are suitable for use in the methods of the present invention.
• a ligand that binds to nicotinic acetylcholine receptors and improves the condition of anxiety by reducing symptoms related to anxiety comprises an organic compound that is a derivative or analogue of tropane.
• the general chemical structure of the tropane derivatives are as follows:
• Ri, R2, R3, R4., R5, R «, and R7 are the same or different and are independently selected from the group consisting of hydrogen, hydroxyl, alkyl, cycloalkyl, alkenyl, alkoxy, aryl, alkylaryl, isoxazole, thiophene, indol, naphthalene, heterocyclic ring, halogen, and amine, as well as their esters and ethers, and Xi, X 2 , and X3 are
• ligands i.e. Class 2 ligands
• ecgonine i.e. Class 2 ligands
• organic compounds including, but not limited to, the following organic compounds: ecgonine;
• RCS-III-202A RCS-III-202A; and analogues and/or derivatives of these compounds.
• the organic compound "ecgonine” has the following chemical structure: Ecgonine
• organic compound "ecgonine methyl ester” has the following chemical structure:
• the organic compound "RTI-4229-70” has the following chemical structure:
• the organic compound "RCS-III-143" has the following chemical structure:
• the organic compound "RCS-III-140A” has the following chemical structure: RCS-III-140A
• the organic compound "RCS-III-218" has the following chemical structure:
• the organic compound "RCS-III-202A” has the following chemical structure:
• a ligand that binds to nicotinic acetylcholine receptors and improves, treats, or prevents anxiety conditions and symptoms, in a subject that is useful in carrying out the methods of the present invention includes one of more of the following cocaine analogs and derivatives:
• Xi, X 2 , and X 3 are independently selected from the group consisting of N, S, O, and C.
• a ligand that binds to nicotinic acetylcholine receptors and improves, treats, or prevents anxiety conditions and symptoms, in a subject that is useful in carrying out the methods of the present invention includes one of more of the following analogs and derivatives of piperidine as follows:
• Ri, R2, R3, R4., R5, and R ⁇ are the same or different and are independently selected from the group consisting of hydrogen, hydroxyl, alkyl, cycloalkyl, alkenyl, alkoxy, aryl, alkylaryl, isoxazole, thiophene, indol, naphthalene, heterocyclic ring, halogen, and amine, as well as their esters and ethers, and Xi, X 2 , and X3 are independently selected from the group consisting of N, S, O, and C.
• a ligand that binds to nicotinic acetylcholine receptors and improves, treats, or prevents anxiety conditions and symptoms, in a subject in accordance with the present invention includes one or more of the following analogues and derivatives of MK-801, but is not dizocilpine.
• analogues and derivatives of MK-801 but is not dizocilpine.
• the general chemical structures of these derivatives are as follows:
• R, Ri, and R2 are the same or different and are independently selected from the group consisting of hydrogen, hydroxyl, alkyl, cycloalkyl, alkenyl, alkoxy, aryl, alkylaryl, isoxazole, thiophene, indol, naphthalene, heterocyclic ring, halogen, and amine, as well as their esters and ethers, and Xi, X 2 , and X3 are independently selected from the group consisting of N, S, O, and C.
• a ligand that binds to nicotinic acetylcholine receptors and improves, treats, or prevents anxiety conditions and symptoms, in a subject in accordance with the present invention includes one of more of the following analogs and derivatives of phencyclidine (PCP), but is not PCP.
• PCP phencyclidine
• the general chemical structures of these derivatives are as follows:
• the present invention relates to a method that improves, treats, or prevents anxiety conditions and symptoms, in a subject, that involves administering to a subject in need of improvement, treatment, and/or prevention of anxiety an aptamer that binds to nicotinic acetylcholine receptors and improves, treats, and prevents anxiety, in a subject.
• RNA aptamers are preferred types of nucleic acid elements that have affinity for and can bind to a target molecule.
• Aptamers typically are generated and identified from a combinatorial library (typically in vitro) wherein a target molecule, generally, although not exclusively, a protein or nucleic acid is used to select from a combinatorial pool of molecules, generally although not exclusively oligonucleotides, those that are capable of binding to the target molecule.
• the selected reagents can be identified as primary aptamers.
• the term "aptamer” includes not only the primary aptamer in its original form, but also secondary aptamers derived from (i.e., created by minimizing and/or modifying the structure of) the primary aptamer. Aptamers, therefore, behave as ligands, binding to their target molecule.
• any method known in the art can be used to identify primary aptamers of any particular target molecule.
• the established in vitro selection and amplification scheme SELEX
• the SELEX scheme is described in detail in U.S. Patent No. 5,270,163 to Gold et al; Ellington and Szostak, "7w Vitro Selection of RNA Molecules that Bind Specific Ligands," Nature 346:818-822 (1990); and Tuerk and Gold, "Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA
• RNA has been established, the RNA molecule can either be prepared synthetically or a DNA construct or an engineered gene capable of encoding such an RNA molecule can be prepared.
• RNA aptamers that can be used in the methods of the present invention, include, but are not limited to, RNA aptamers that have the consensus sequences:
• SEQ ID NO: 1 i.e., ACCG
• SEQ ID NO: 2 i.e., UCCG
• SEQ ID NO: 3 i.e., UUUACCG
• SEQ ID NO: 4 i.e., UUCACCG
• SEQ ID NO: 5 i.e., UUCACCGUAAGG
• SEQ ID NO:6 i.e., AUCACCGUAAGG (see Aptamer B5)
• SEQ ID NO:7 i.e., UUUACCGUAAGG (see Aptamer B 15)
• SEQ ID NO:8 i.e.,
• UUUUCCGUAAGG see Aptamer B19
• SEQ ID NO:9 i.e., UUUACCGUAAGG (see Aptamer B27)
• SEQ ID NO: 10 i.e., AUCACCGUAAGG (see Aptamer B28)
• SEQ ID NO: 11 i.e., UCCACCGUAGAU (see Aptamer B36)
• SEQ ID NO: 12 i.e.,
• AUCACCGUAAGG see Aptamer B44
• SEQ ID NO: 13 i.e., UUUACCGUAAGG (see Aptamer B55)
• SEQ ID NO: 14 i.e., UCCACCGUAAGA (see Aptamer B59)
• SEQ ID NO: 15 i.e., UCCACCGUAAGA (see Aptamer B61)
• SEQ ID NO: 16 i.e.,
• UUUACCGUAAGG see Aptamer B64
• SEQ ID NO: 17 i.e., UUUACCGUAAGG (see Aptamer B65)
• SEQ ID NO: 18 i.e., UUUACCGUAAGG (see Aptamer B69)
• SEQ ID NO: 19 i.e., UCCACCGUAAGA (see Aptamer B76)
• SEQ ID NO:20 i.e.,
• UUUUCCGUAAGG see Aptamer B78
• SEQ ID NO:21 i.e., UCCACCGUAAGA (see Aptamer B 108)
• SEQ ID NO:22 i.e., UUUACCGUAAGG (see Aptamer Bl 11)
• SEQ ID NO:23 i.e., AUCACCGUAAGG (see Aptamer B124)
• SEQ ID NO: 66 i.e., GAAAG
• SEQ ID NO: 88 i.e., GUUAAU
• RNA aptamers that can be used in the methods of the present invention, include, but are not limited to, RNA aptamers having nucleotide sequences:
• SEQ ID NO:24 (Aptamer B5), SEQ ID NO:25 (Aptamer B 15), SEQ ID NO:26 (Aptamer B 19), SEQ ID NO:27 (Aptamer B27), SEQ ID NO:28 (Aptamer B28), SEQ ID NO:29 (Aptamer B36), SEQ ID NO:30 (Aptamer B44), SEQ ID NO:31 (Aptamer B55), SEQ ID NO:32 (Aptamer B59), SEQ ID NO:33 (Aptamer B61), SEQ ID NO:34 (Aptamer B64), SEQ ID NO:35 (Aptamer B65), SEQ ID NO:36 (Aptamer B69), SEQ ID NO:37 (Aptamer B76), SEQ ID NO:38 (Aptamer B78), SEQ ID NO:39
• SEQ ID NO:42 (Aptamer 01), SEQ ID NO:43 (Aptamer 05), SEQ ID NO:44 (Aptamer 06), SEQ ID NO:45 (Aptamer 07), SEQ ID NO:46 (Aptamer 09), SEQ ID NO:47 (Aptamer 11), SEQ ID NO:48 (Aptamer 13), SEQ ID NO:49 (Aptamer 14), SEQ ID NO:50 (Aptamer 16), SEQ ID NO:51 (Aptamer 18), SEQ ID NO:52 (Aptamer 19), SEQ ID NO:53 (Aptamer 20,21), and/or SEQ ID NO:54 (Aptamer 22);
• modified aptamers having improved properties such as decreased size, enhanced stability, or enhanced binding affinity.
• modifications of aptamer sequences include adding, deleting or substituting nucleotide residues, and/or chemically modifying one or more residues.
• Methods for producing such modified aptamers are known in the art and described in, e.g., U.S. Patent Nos. 5,817,785 to Gold et al., and 5,958,691 to Wolfgang et al, which are hereby incorporated by reference in their entirety.
• Chemically modified aptamers include those containing one or more modified bases.
• modified pyrimidine bases may have substitutions of the general formula 5'-X and/or 2'-Y
• a modified purine bases may have modifications of the general formula 8'-X and/or 2'-Y.
• the group X includes the halogens I, Br, CI, or an azide or amino group.
• the group Y includes an amino group, fluorine, or a methoxy group. Other functional substitutions that would serve the same function may also be included.
• the aptamers of the present invention may have one or more X-modified bases, or one or more Y-modified bases, or a combination of X- and Y-modified bases.
• the present invention encompasses derivatives of these substituted pyrimidines and purines such as 5'-triphosphates, and 5'-dimethoxytrityl, 3'-beta-cyanoethyl, N,N-diisopropyl phosphoramidites with isobutyryl protected bases in the case of adenosine and guanosine, or acyl protection in the case of cytosine.
• these substituted pyrimidines and purines such as 5'-triphosphates, and 5'-dimethoxytrityl, 3'-beta-cyanoethyl, N,N-diisopropyl phosphoramidites with isobutyryl protected bases in the case of adenosine and guanosine, or acyl protection in the case of cytosine.
• nucleotide analogs modified at the 5 and 2' positions, including 5-(3-aminoallyl)uridine triphosphate (5-AA-UTP), 5-(3-aminoallyl) deoxyuridine triphosphate (5-AA-dUTP), 5 -fluorescein- 12 -uridine triphosphate (5-F-12- UTP), 5 -digoxygenin- 11 -uridine triphosphate (5-Dig-l 1-UTP), 5-bromouridine triphosphate (5-Br-UTP), 2'-amino-uridine triphosphate (2'-NH2-UTP) and 2'-amino- cytidine triphosphate (2'- NH 2 -CTP), 2'-fluoro-cytidine triphosphate (2'-F-CTP), and 2'- fluoro -uridine triphosphate (2'-F-UTP).
• nucleotide analogs modified at the 5 and 2' positions, including 5-(3-aminoallyl)uridine tri
• the aptamers may also be modified by capping at the 3' and 5' end.
• the aptamer can be modified by adding to an end a polyethyleneglycol, amino acid, peptide, inverted dT, nucleic acid, nucleosides, myristoyl, lithocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl, other lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, toxin, enzymes, radioactive substance, biotin and the like.
• U.S. Patent Publication No. 2005/0096290 to Adamis et al. and U.S. Patent No. 5,660,985 to Wolfgang et al which are hereby incorporated by reference in their entirety.
• the nicotinic acetylcholine receptor ligands that improve, prevent, or treat anxiety and anxiety symptoms of the present invention can be administered orally, parenterally, for example, subcutaneous ly, intravenously, intramuscularly,
• ligands may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
• the nicotinic acetylcholine receptor ligands that improve, prevent, or treat anxiety and anxiety symptoms of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
• an inert diluent or with an assimilable edible carrier
• the anxiety enhancing ligands including compounds and aptamers of the present invention may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
• Such compositions and preparations should contain at least 0.1% of active compound.
• the percentage of anxiety-treatment enhancing ligand of the present invention in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
• the concentration of nicotinic acetylcholine receptor ligand in such therapeutically useful composition is such that a suitable dosage will be obtained.
• Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 and 250 mg of one or more cognition enhancing ligands of the present invention.
• the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
• a binder such as gum tragacanth, acacia, corn starch, or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid
• a lubricant such as magnesium stearate
• a sweetening agent such as sucrose, lactose, or saccharin.
• a liquid carrier such as a fatty oil.
• tablets may be coated with shellac, sugar, or both.
• a syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
• the nicotinic acetylcholine receptor ligands that improve, prevent, or treat anxiety and anxiety symptoms of the present invention may also be administered parenterally.
• Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
• Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
• water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy use in syringes exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• the nicotinic acetylcholine receptor ligands that improve, prevent, or treat anxiety and/or anxiety symptoms of the present invention may also be administered directly to the airways in the form of an aerosol.
• the compounds of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
• suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
• the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
• IP intraperitoneal
• EME ecgonine methyl ester
• Thigmotaxis is a commonly used index or measure of anxiety in animals.
• thigmotaxis is noted by the amount of time a rat swims along the wall of a water tank, indicating anxiety and a 'desire' or expectation to find an exit along the edge. The longer the time spent along the edge, the more anxiety is present in the animal.
• Thigmotaxis in rats can be studied as a function of age, disease, drug treatment etc., using the Morris water maze test.
• a single rat is place in a circular water bath/swimming pool equipped with a visible or submerged platform, and the time and route taken to reach the platform for the rat is measured and recorded electronically.
• the internal surface of the wall is decorated with images of symbols of various types such that the rat can accumulate knowledge of the geometry of the pool and when the platform is found, how to subsequently reach the platform as expeditiously as possible. Once on the platform the rat tends to stay there, as this is a state preferable to swimming.
• Time to reach the platform, distance traveled, speed etc., are recorded for the observer electronically and a permanent image is produced showing the route taken.
• the whole exercise takes a few seconds in normal circumstances.
• the rats are trained over a period of time to a steady-state level of competence, and a probe test then measures how well the rats have learned and remembered the position of the platform, which can be visible or submerged in different versions of the test.
• the rats can be studied during the training program, so that the investigator can gain insight into the speed of, for example, learning.
• learning can be efficient, but then changing the position of the platform can present a whole new challenge, such that the investigator can discriminate between drug effects on the learning process itself and on the ability to perform better in the state created by the learning.
• Three examples are here presented to illustrate the phenomenon of thigmotaxis as affected by EME treatment, a Class 2 nicotinic acetylcholine receptor ligand.
• Figure 2A shows a Morris water maze trace of a young control rat swimming in the bath for approximately 30 seconds searching for the platform. The trace indicates that the animal covered various quadrants of the pool quite easily, without evidence of anxiety, demonstrating moderately uniform attention to the different areas of the pool, not finding the platform until the last few seconds of the test.
• Figure 2B shows a Morris water maze trace of an aged and rather anxious rat swimming almost entirely at the perimeter of the water bath, finding the platform briefly at one stage but not staying there as if not certain in his anxious state that staying on the platform would be a good idea.
• Figure 2C shows a Morris water maze trace of the same rat shown in Figure 2B, following ten days of treatment with EME. Following treatment the rat learned how to find the platform, taking his cues from the wall markings, and lost his state of anxiety previously illustrated by the hugging of the walls phenomenon and the hesitancy to stay on the platform when he had found it.
• FIG. 3 shows mean results in the four groups of rats, including those treated with repeat doses of EME for an extended period of time.
• the groups were young controls, aged drug- free controls, aged treated with 3 mg/kg of EME, and aged treated with 10 mg/kg of EME.
• the young rats showed very little thigmotaxis, and recovered from what occurred quite quickly.
• the aged rats started from the same baseline as the young rats, and recorded the highest overall level of thigmotaxis of the four groups.
• a new challenge led to reinstatement of impaired performance and a new searching and finding cycle, even with drug on board at the time of the change in platform position.
• the young rats showed little or no thigmotaxis, reaching maximum learned performance by the third day.
• the untreated aged rats learned more slowly, gradually showing improved performance over the 10 days (first trial) and the five days (second trial) reaching a maximum level of performance about one half that of the young rats.
• the drug-treated aged rats showed faster learning, with the performance of rats receiving 10 mg/kg EME coming close to that of the young controls by the tenth day.
• a repeat test showed the same results as Figure 4, particularly demonstrating the acute response to the new challenge and a new cycle of searching and learning.
• pooled data are displayed with standard errors of means (SEMs) calculated. Pooling of such large volumes of data is generally used to only obtain guidance concerning the appropriate testing strategy.
• SEMs standard errors of means

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