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WO1994009765A1 - Interactions fonctionnelles entre le s-100b glial et les neurones serotoninergiques du systeme nerveux central - Google Patents

Interactions fonctionnelles entre le s-100b glial et les neurones serotoninergiques du systeme nerveux central Download PDF

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Publication number
WO1994009765A1
WO1994009765A1 PCT/US1993/010095 US9310095W WO9409765A1 WO 1994009765 A1 WO1994009765 A1 WO 1994009765A1 US 9310095 W US9310095 W US 9310095W WO 9409765 A1 WO9409765 A1 WO 9409765A1
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receptor
antibody
neurons
subject
serotonergic
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PCT/US1993/010095
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English (en)
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Efrain C. Azmitia
Patricia M. Whitaker-Azmitia
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New York University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
    • 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
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention in the field of neuroscience and medicine relates to the discovery and use of a cortical growth factor S-100 B as trophic for cortical and serotonergic neurons in the brain, as well as the use of 5-HT----A agonists, S-100 B and derivatives thereof, to induce corticol or serotonergic growth, 10 stimulation or regeneration, for therapeutic and/or diagnostic applications, in vivo, in si tu and/or in vi tro .
  • the present invention relates to the use of 5-H7-- 1A antagonists, also including peptides corresponding to functional domains of 5-HT--jA receptors, and antibodies thereto, which up regulate 15 central acyonergic neurons, which subsequent stimulation is enhanced or provided by such up regulation.
  • Serotonergic neurons which release serotonin (5- hydroxytryptamine, 5-HT) as a neurotransmitter, play a key role 20 in the general maturation of the brain. Changes in the innervation density of serotonergic nerve fiberj would be expected to induce changes in the maturation of "target" neurons with which the serotonergic fibers communicate.
  • Serotonin neurons have been shown to regulate their own development, i.e. to "autoregulate" (Whitaker-Azmitia et 35 al., Neuro ⁇ ci . Lett. 67:307-312 (1986)), due in part to release of growth factors by stimulation of 5-HT 1A recepuors on astrocytes (Whitaker-Azmitia et al., J. Neurochem. 46:1186-91 (1986)). Glial cells, such as astrocytes, have 5-HT receptors (Whitaker et al. , ibid. ) .
  • Nerve growth factor appears to act as a CNS cholinergic growth factor (Hefti, J. Neurosci . 6:2155-2162 (1986)).
  • Epidermal growth factor (EGF) has trophic effects on neuron-like PC-12 cells (Leonard et al. , Mol . Cell . Biol . 7:3156-3167 (1987); Isobe et al. , J “ . Neurochem. 43:1494-1496 (1984) ) .
  • Insulin has been shown to mediate growth of cultured fetal neurons (Heindenreich et al. , Endocrinology 125:1451-1457 (1989)).
  • the protein S-100 B/ which is composed of two ⁇ - subunits, stimulates neurite extension in cultured chick cortical neurons, hence its designation as a "cortical" growth factor (Kligman et al. , Proc. Natl . Acad. Sci . U. S.A . 82:7136- 7139 (1985) ) .
  • Calmodulin has been shown to have substantial structural homology and a similar Ca 2+ binding profile to S-100 B (Isobe et al. , Endocrinology 125:1451-1457 (1989)).
  • S-100 A family of proteins named S-100 was first isolated nearly 25 years ago (Moore, Biochem. Biophys . Res. Commun . 19:739-742 (1965)). The member of this family designated S-100 B was previously known to promote neurite extension in vitro, in chick embryo cultures (Kligman et al., Proc. Natl . Acad. Sci . USA 82:7136-39 (1985)). S-100 production was stimulated in cultures of the rat astroglioma line, C6, by dibutyryl cyclic AMP (Labourdette et al., Biochem. Biophys . Res. Comm. 96:1702-09 (1965)). Furthermore, S-100 may be releasable from brain tissue (Shashoua et al. , J " . Neurochem. 42:1536-41 (1984)).
  • NGF neurotrophic factor
  • a cDNA for the 5-HT 1A receptor from the rat has been cloned and shown to have a coding region consisting of 1266 nucleotides corresponding to the 422 amino acids! of 5-HT 1A (Albert et al, J. Biol . Chem. 265:528 1990).
  • the 5-HT 1A receptor from rat has seven transmembrane domains, a large third cytoplasmic loop and is 89% homologous with the human gene.
  • the rat 5-HT 1A receptor encoding mRNA tissue distribution showed high levels in spectrum, hippocampus, thalamus, amygdala olfactory bulb, mesencephalon, medulla and hypothalamus.
  • S- 100 B is a protein released upon stimulation of their 5-HT JA receptors, which stimulation has further been found to promote the growth of central serotonergic neurons and/or cortical neurons.
  • the present invention also relates to methods for modulating S100 B effects in vi tro, in vivo and in si tu, as well as to diagnostic and/or therapeutic methods involving serotonergic neuronal growth and/or maintenance, through such modulation of S100 B .
  • the present invention is also directed, in one aspect, to a method for stimulating the production of S-100 B in a subject, comprising administering a S100 B stimulating effective amount of an agonist acting on the 5-HT 1A receptor, by the use of at least one 5-HT-- 1A agonist compound or anti-idiotype antibody to 5-HT-- 1A receptors which has the in vivo effect of stimulating 5-HT 1A receptors.
  • Non-limiting examples of 5-HT-- 1A agonists useful in the present invention may include 5-hydroxytryptamine, 5-methoxytryptamine, buspirone, 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-B AT) , ipsaspirone, gepirone, SM ⁇ '3997, lysergic acid, ipsapirone, diethylamide, and 5-HT-- 1A agonistic antibodies.
  • the stimulation of 5-HT-- IA receptors may be made subsequent to up regulation of such receptors, which prior up regulation increases the stimulatory effect on cortical and/or serotonergic neurons, preferably serotonergic neurons, which prior up regulation can be accomplished according to known method steps, as known to those skilled in the art, based on the teachings and guidance presented herein.
  • Stimulation of S-100 B or 5-HT-- 1A receptor stimulation in a subject may have the therapeutic or diagnostic effect of causing serotonergic neuron growth and/or stimulation, which may be suitable for treatment and/or diagnosis of at least one diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-sleep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers disease, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotonin production developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction.
  • the present invention is further directed to a method for inducing the growth and/or stimulation of central serotonergic neurons or serotonin release in a subject, comprising administering to the subject an effective amount of S-100 B , a functional derivative or analog thereof, or an agonist acting at the 5-HT 1A receptor, such as those described above and including nti-idiotype antibodies to at least one functional domain of a 5-HT-- 1A receptor, from a mammal, preferably a human.
  • the present invention also includes a method for inducing the growth and/or stimulation of central serotonergic neurons or serotonin, in vitro or in vivo by contacting the neurons with S100 B , a 5-HT-- 1A agonistic antibody, or a functional derivative or analog thereof.
  • a method for inducing the growth and/or stimulation of central serotonergic neurons or serotonin in vitro or in vivo by contacting the neurons with S100 B , a 5-HT-- 1A agonistic antibody, or a functional derivative or analog thereof.
  • S100 B a 5-HT-- 1A agonistic antibody
  • a functional derivative or analog thereof for treatment of diseases associated with dysregulation of serotonergic neurons.
  • the present invention involves a method for inhibiting the growth of central serotonergic neurons, comprising contacting the neurons with an effective amount of an inhibitor of S-100 B production or action.
  • the inhibitor may be an antibody specific for S-100 B or a 5-HT 1A receptor antagonist, such as the non-limiting examples of spiperone and spiroxatine, or an 5-HT-- 1A receptor peptide corresponding to a functional domain of a 5-HT-- 1A receptor, or antibodies thereto, such as anti-peptide antibodies.
  • the present invention also relates to a method for treating a disease and/or pathology associated with decreased central serotonergic innervation or activity including decreased serotonin levels in a mammalian, preferably human, subject, by administering a serotonergic stimulating effective amount of S- 100 B , a functional derivative thereof, or a 5-HT ]A agonist including, but not limited to an antibody to a functional domain of a 5-HT-- 1A receptor.
  • Diseases for which this method is useful include diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-fileep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotonin production developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction.
  • the present invention is also directed to a method for treating a disease associated with increased. central serotonergic innervation or activity in a subject, comprising administering an effective amount of an inhibitor of S-100 B production or action.
  • Such inhibitors include an antibody specific for S-100 B 5-HT 1A receptors, and/or a 5-HT ⁇ A receptor antagonist.
  • the invention is directed to a method for stimulating serotonergic neuronal growth and/or stimulation in a subject having Alzheimer's disease comprising the steps of: (a) up-regulating the expression of 5-HT-- 1A receptors on astroglial cells in the brain of the subject; and then (b) stimulating the induction of the release of S-100 B in the subject according to the methods described above, thereby stimulating the cortical neuronal growth.
  • Figure 3 is a histogram showing a morpnometric analysis of the total neurite length for individual 5-HT- immunoreactive neurons after 30 h of stimulation. The bars represent the mean + S.E.M. for 10 neurons in each well (number shown under bars) .
  • Figure 4 is a graph showing effects of native bovine S-100 and of media from astroglial cells stimulated with the selective 5-HT ⁇ A agonist ipsapirone (GCM-IPS)* on the growth of serotonergic neurons in culture as determined by selective uptake of 3 H-serotonin. Hatched bars indicate the effects of S- 100 and GCM-IPS in the presence of an antibody o S-100 (final dilution 1/10,000) . Each bar represents the mean ⁇ S.E.M. of 4 cultures, derived from different litters. S-lO ⁇ , GCM-IPS and the antibody were all added at time of plating and the grpwth assessed 3 days later.
  • Figure 5 is a pictorial representation of antipeptide 5-HT-- 1A receptor antibody binding to a hippocampus in midbrain section of a rat brain at 13500 times wherein tr.3 label is associated with microtubules (MTB) and also bound along the outer plasma membrane (PLMB) neurons.
  • MTB microtubules
  • PLMB outer plasma membrane
  • Figure 6 shows a schematic diagram of the 5-HT-- JA receptor.
  • Figure 7 shows a graphical representation of a purified synthetic peptide corresponding to a portion of a functional domain of a 5-HT-- 1A receptor.
  • Figure 8 shows a graphical representation of radio- labeled anti-goat anti-rabbit Ig fragments to antipeptide antibodies of the present invention wherein labeling radioactivity it various antibody dilutions.
  • the present invention relates to the discovery that a protein designated S-100 B or functional derivatives and/or analogs thereof, is a serotonergic specific growth factor (SGF) that is also induced by serotonin. Since this protein has no effect on cholinergic and noradrenergic neurons, nor on cells in the peripheral nervous system, S-100 B , or derivatives or analogs thereof, are specific for modulation of central serotonergic nerves.
  • SGF serotonergic specific growth factor
  • the present invention also relates to the discovery that S-100 B is a growth factor released in response to 5-HT 1A receptor stimulation as well as by serotonin. Serotonin neurons have been shown to autoregulate their own development. The present inventors first discovered that this autoregulatory circuit involves the release of a growth factor or factors induced by stimulation of 5-HT 1A receptors in vivo, such as on astrocytes. Therefore, the present invention is directed in one aspect to the use of 5-HT ]A agonists or antagonists as therapeutics, acting via the regulation and/or modulation of S- 100 B production and/or release from astroglial cells and/or serotonergic neurons.
  • the present invention is also directed to methods involving the use of the S-100 B protein, which is a dimer of two ⁇ chains, and is found exclusively in the brain, in contrast to ⁇ - o ⁇ or ot- ⁇ dimers, which are also found outside the brain. Also included within the scope of the present invention are functional derivatives of the S-100 B protein.
  • An “analog” of S-100 B refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • модород By “functional derivative” is meant a “fragment, " “variant, “ or “chemical derivative” of S-100 B , which terms are defined below.
  • a functional derivative retains at least a portion of the function of the S-100 B which permits its utility in accordance with the present invention, namely serotonergic or cortical growth factor activity.
  • a “fragment” of the S-100 B refers to any subset of the molecule, or of the ⁇ chain, such as a shorter peptide.
  • a “variant” of the S-100 B refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, or by recombinant DNA technology, using well known method steps. Amino acid sequence variants of the S-100 B molecule can also be prepared by mutations in the DNA. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence.
  • deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity, without placing the sequence out of reading frame and preferably not creating complementary regions that could produce secondary mRNA structure (see EP Patent Application Publication No. 75,444) .
  • these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding the S-100 B molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant ceil culture.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analog.
  • variants have greater than 80% homology with the corresponding S100 B proteins or fragments, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, while maintaining at least some HT-5 JA receptor modulating activity.
  • a "chemical derivative" of S-100 B contains additional chemical moieties not normally a part of the protein or peptide.
  • Covalent modifications of the* protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • salts of the proteins and peptides of the invention are also included in the scope of the invention. As used herein, the term “salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein or peptide molecule.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
  • agonist is intended any chemical or biological substance capable of binding to a particular receptor, such as the 5-HT 1A receptor or binding site thereof, according to the present invention, which binding stimulates a biological response associated with the receptor.
  • the term is intended to include an endogenous molecule which exerts its physiological action by receptor binding and triggering of a signal to a cell, as well as an exogenous agent which mimics the action of such an endogenous agonist.
  • a receptor is linked to a second messenger system that signals a positive response, such as, for example, the increased production and/or secretion of a protein growth factor, such as S100 B , or analog or functional derivative thereof, an agonist will induce production and/or secretion of the growth factor.
  • a receptor is linked to a second messenger system which signals a negative response, such as a termination of cell growth, an agonist for that receptor will inhibit cell growth.
  • Agonists for 5-HT 1A receptors include, but are not limited to, 5-hydroxytryptamine (serotonin) , 5- methoxytryptamine, buspirone (U.S. Patent 3,717,634) , 8- hydroxydipropylamineotetralin, ipsaspirone (EPO Publication 129,128A), gepirone (U.S. Patent 4,423,049) , SM23997 (U.S. Patent 4,507,303) , MDL 72832 (U.S. Patent 4,612.312) ipsapirone, lysergic acid diethylamide and anti-idiotype ancibodies to one or more functional domains of a 5-HT-- 1A receptor.
  • newer polycyclic aryl- and heteroarylpipera- zinyl imides with 5-HT 1A -binding and activating properties such as WY-47,846 (cpd. 34) and other disclosed in Abou-Gharbia et al. , J. Med. Chem. 31:1382-1392 (19?8) , may be useful in the prese.'i invention, which references are hereby entirely incorporated by reference.
  • an antibody preferably a monoclonal antibody (MAb) to the 5-HT 1A receptor which by virtue of its epitope specificity stimulates a response, rather than inhibiting the binding of an agonist, termed an "agonistic antibody, " is also an agonist as intended herein.
  • MAb monoclonal antibody
  • antagonist is intended a substance which is itself devoid of intrinsic pharmacolog-. activity and stimulates no biological response when bound to a receptor, but has the capacity to bind to a receptor and thereby inhibit binding of, or action of, an agonist.
  • antagonists act by competing for agonist binding to a receptor.
  • Antagonists for 5-HTj A receptors are known in the art and include, but are not limited to, spiperone and spiroxatine.
  • an antibody specific for the 5-HT, A receptor which does not have agonist activity, but inhibits bi::ding or action of an agonist is also an antagonist, as intendRd herein.
  • newer polycyclic aryl- and heteroaryl-piperazinyl imides with 5-HT 1A -binding properties which would inhibit binding of endogenous agonists may be useful as antagonists in the present invention.
  • a 5-HT-- 1A peptide, as described herein, or a 5-HT-- 1A antagonistic antibody to such pept.i.des, preferably a monoclonal antibody (MAb) to the 5-HT 1A receptor which, by virtue of its epitope specificity, inhibits the binding of a 5- HT-- 1A agonist, termed an "antagonistic antibody,” is also an antagonist as intended herein.
  • the present invention is intended to encompass additional 5-HT 1A receptor antagonists, routinely obtainable according to the present invention, based on the teaching and guidance presented herein without undue experimentation.
  • peptides corresponding to portions of functional or other domains, such as transmembrane domains, of 5-HT-- 1A receptors have also unexpectedly been discovered to act as 5-HT-- 1A receptor antagonists, as do 5-HT-- 1A receptor antagonists, as described herein.
  • Antibodies of the present invention are those which are specific for, and interact with, S-100 B , funrational derivatives or analogs thereof, or with 5-HT-- 1A receptors and modulate the action of the S-100 B -serotonergic neuron autoregulatory system, e.g., as 5-HT-- 1A receptor agonists or antagonists.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (mAbs) , chimeric antibodies, and anti-idiotypic (anti-Id) antibodies or fragments, analogs or derivatives thereof.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from -he sera of animals immunized with an antigen.
  • a "5-HT-- 1A antibody” refers to an antibody, as described herein, which binds or associates with a 5-HT-- 1A receptor and has the in vivo biological activity of stimulating or inhibiting the 5-HT-- 1A receptor to have the effect of inducing or inhibiting, respectively, serotonergic and/or cortical neuron growth and/or stimulation, which effect may be mediated by the release of S- 100 B , and wherein the modulation of central acyonergic neurons is preferred.
  • Such antagonistic 5-HT-- 1A antibodies of the present invention are preferably generated against a synthetic peptide corresponding to a functional domain of a 5-HT 1A receptor, according to the following criteria.
  • the receptor is homologous to the beta-adrenergic receptor family and many of the various segments of the 5-HT 1A receptor can be inferred from the extensive work with the ⁇ -adrenergic receptor.
  • the agonist binding site consists of a least two aspartate (asp) residues within the 2nd and 3rd transmembrane regions (Dohlman et al. Biochemistry 26:2657-2663 (1987). Asp residues (#82 and 116) exist in a similar site in the 5-HT 1A receptor ( Figure 6) .
  • a histidine (His) in the 3rd transmembrane site (#126) would provide the needed positive charge for 5-HT binding.
  • the third cytoplasmic loop is believed to be the site of interaction with the G-proteins in the cytoplasm for regulation of the second messenger systems (Kobilka et al. Nature 329:205-230 (1988)). Therefore, peptide sequences can be selected indicate the anatomical location of various segments of the full molecule and determine the cellular distribution of particular functional regions.
  • domains vere selected in the 2nd external loop (S1A-170) and in the 3rd cytoplasmic loop (S1A-258) (Fig. 6) .
  • the antigenic sites on a peptide can be approximated based on the hydrophilicity score which assumes that the greater the local hydrophilicity, the more antigenic the sequence (Hopp, T.P. Proc. Natl. Acad. Sci. USA 8:3824-3828 (1981)).
  • This measure assigns a numerical value to the various amino-acids; for example K, R, D and E have a value of +3.00, W has a value of -3.4, and G and P have a value of 0. the calculated window average at a residue is calculated across 6 residues.
  • the hydrophilicity score for A1A170 is shown in Table I.
  • the net charge of a peptide sequence should be near neutrality. If the molecule is too highly charged it will present problems during the purification procedure after the peptide is synthesized. If the net charge is highly basic or acidic a cation or anion exchange resin can be used. Bio-rad AG-50 resin has been successfully used for very basic peptides. However, strong deviations from neutrality is also a problem during the attachment to KLB which should proceed at neutral pH (see below) .
  • S1A-170 has 6 charged residues and a net -2 charge while S1A-258 has 5 charged residues and a net + 1 charge.
  • Amino Acid length The sequen e for an ideal peptide for antibody formation should have 15-20 amino acids. A strand of 6 amino acids is the lower limit for a recognition site whole more than 20 presents some additional problems with the synthesis and structural considerations (Harlow, E. et al. Antibodies: A Laboratory Manual Cold Spring Harbor Press (1988) ) .
  • both S1A-170 and S1A-258 have 17 residues.
  • Phosphorylation and glycosylation sites A protein molecule has many possible phosphorylation and glycosylation sites. These sites should be avoided in choosing a sequence unless a particular confirmation is sought. Antibodies have ueen raised against phosphoryla*'2d sequences but these antibodies have altered affinity for the un-phosphorylated site (See Czernik et al. Method Enzymol 201:264-283 (1991) . Furthermore, a phosphorylated segment of the molecule often confers allosteric changes in the protein structure which may reduce the affinity for the peptide segment artificially produced. It can be appreciated, that sites adjacent to modified sites may be less desirable for the same reasons.
  • glycosylation sites are located on the (ASN, N) residues at positions 10, 11 and 24.
  • Three potr-'.ntial protein kinase C phosphorylation sites are located at 147-152, 227-232 and 341-345 and one additional phosphorylation site 251-253 (El Mestikawy et al, Neurochem. Res. 16:1-10 (1991)).
  • Cystein-.* (Cys, C) residues are commonly involved in disulfide bridges. For this reason it is advisable to avoid a Cys residue in the middle of a peptide sequence.
  • Cysteine residues in the 5-HT 1A receptor 6 in the transmembrane regions, 3 in the 3rd cytoplasmic loop, four in the three extracellular loop and two in the C-terminal cytoplasmic tail.
  • cysteines in extracellular loop 1 and 2 have been proposed to form a disulfide link in the /3 2 -adrenergic receptor (Dchlman et al. Biochemistry 26: (1987)). Similar cysteines ex: : .st in the 5-HT 1A receptor ( Figure 6) .
  • a terminal cysteine residue in order to bind to KLH protein (Harlow, et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press (1988) .
  • This can be either at the C- or N-terminus of the peptide depending on how the peptide is predicted to be exposed in the molecule. For instance, if the desired sequence is at the N-terminal end of the protein, then the cysteine should be placed at the C-terminal end of the peptide. In this way, the N-terminal end will be exposed after its attachment to the carrier protein.
  • both peptides have an N- terminal Cys ut S1A-258 also contains a Cys in the center of the peptide at position 266.
  • the present invention is also related to the production, by chemical synthesis or recombinant DNA technology, of 5-HT-- 1A receptor peptides, preferably as small as possible while still retaining sufficiently high affinity or interaction with G-protein coupled receptors to modulate, such as to inhibit binding to such receptors by 5-HT-- 1A receptor ligands.
  • 5-HT-- 1A receptor peptides of the present invention may include 5-10 to 50-150 amino acid fragments, consensus sequences or substitution sequences of 5-HT-- 1A receptors, including, but not limited to serotonin receptors (5-HT) , cytomegalovirus 5-HT-- IA receptors, endothelial cell 5-HT-- 1A receptors, testi 5-HT-- 1A receptors, and thoracic aorta 5-HT-- IA receptors, and homologs thereof having a homology of at least
  • a "5-HT-- 1A receptor peptide" of the present invention includes polypeptides having a "5-HT-- ⁇ A receptor amino acid sequence" which substantially corresponds to at least one 4 to 50 amino acid fragment and/or consensus sequence of a known 5-HT-- 1A receptor or group of 5-HT-- 1A receptors, wherein the 5-HT--- IA receptor peptide has homology of at least 80%, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology, while maintaining 5-HT-- ⁇ A receptor modulating activity, wherein a 5- HT-- 1A receptor peptide of
  • a 5- HT-- 1A receptor peptide of the present invention substantially corresponds to a transmembrane domain of a 5-HT-- 1A receptor or group of 5-HT-- 1A receptors as a consensus sequence.
  • 5-HT-- 1A receptor peptides wherein the 5-HT-- 1A receptor amino acid sequence is 4-10 to 50 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids, or any range therein.
  • An amino acid or nucleic acid sequence of a 5-HT-- j * A receptor peptide of the present invention is said to "substantially correspond" to another amino aci£ or nucleic acid sequence, respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one 5-HT-- 1A receptor transmembrane domain, or which may be synergistic when two or more transmembrane domains, consensus sequences or hoirologs thereof are present.
  • substantially corresponding sequences of 5-HT-- 1A receptor peptides include conservative amino acid or nucleotide substitutions, or degenerate nucleotide codon substitutions wherein individual amino acid or nucleotide substitutions are well Known in the art.
  • substantially corresponding refers to 5-HT-- 1A receptor peptides having amino acid sequences having at least 80% homology or identity to an amino acid sequence of a human 5-HT- ⁇ ⁇ A receptor, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology or identity.
  • 5-HT-- ⁇ A receptor peptides of the present invention include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • For a detailed description of protein chemistry and structure see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T.E., Proteins : Structure and Molecular Properties , W.H. Freeman & ⁇ Co., San Francisco, 1983, which are hereby incorporated by reference.
  • nucleotide sequence substitutions such as codon preferences, see Ausubel et al, supra, at ⁇ A.l.l-A.1.2
  • Conservative substitutions of a 5-HT-- 1A receptor peptide of the present invention includes a variant wherein at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid.
  • Such substitutions preferably are made in accordance with the following list as presented in Table IV, which substitutions may be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule, while maintaining the receptor binding, inhibiting or mimicking biological activity, as determined by known 5-HT-- 1A receptor receptor activity assays.
  • IA receptor peptides of the present invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue inserted in its place according to the following Table V.
  • the types of substitutions which may be made in the protein or peptide molecule of the ⁇ resent invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al., supra and Figs. 3-9 of Creighton, supra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: TABLE V
  • Conservative amino acid substitutions according to the present invention are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. a-helix or ⁇ -sheet, as well as changes in physiological activity, e.g. in receptor binding assays.
  • substitution, deletion, or insertion when the exact effect of the substitution, deletion, or insertion is to be confirmed one skilled in the art will appreciate that the effect of the substitution or substitutions will be evaluated by routine screening assays, either immunoassays or bioassays to confirm biological activity, such as receptor binding or modulation of ligand binding to the corresponding 5-HT-- 1A receptor.
  • a substituted polypeptide typically is made by site-speci ic mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a chemically derivatized column or immobilized membranes or hollow fibers (to absorb the mutant by binding to at least one epitope) .
  • a preferred use of this invention is the production, by chemical or recombinant DNA technology, of 5-HT-- 1A receptor peptides, preferably as -small as possible while still retaining sufficiently high affinity for binding to, or association with, 5- HT-- 1A receptors.
  • 5-HT-- 1A receptor peptides including smaller fragments or variants of such transmembrane domains, one skilled in the art, using known binding and inhibition assays, can readily identify the 5-HT--- IA receptor peptides capable of binding minimizing or modulating G-protein coupled receptors using known methods.
  • 5-HT--, A receptor peptides may include consensus sequences and/or fragments of at least one of transmembrane domain 1-7 of one or more 5-HT-- 1A receptors, which 5-HT-- 1A receptor peptides do not occur naturally, and/or which are provided in an isolated and/or purified form not found in nature.
  • Consensus peptides of 5-HT-- 1A receptor peptides of the present invention may include peptides which are distinct from known 5-HT-- 1A receptor sequences in critical structural features, but which are derived from consensus sequences of homologous 5-HT-- 1A receptor transmembrane domains 1-7.
  • consensus peptides may be derived by molecular modeling, optionalJy combined with hydrophobicity analysis and/or fitting to model helices, as non-limiting examples. Such modeling can be accomplished according to known method steps using known modeling algorithms, such as, but not limited to, ECEPP, INSIGHT, DISCOVER, CHEM-DRAW, AMBER, FRODO and CHEM-X.
  • Sue 1 algorithms compare transmembrane domains between related G-protein coupled receptors, determine probable energ - miminized structures and define alternative consensus polypeptide fragments.
  • Such consensus peptides or fragments of 5-HT-- 1A receptors may then be synthesized or produced recombinantly, in order to provide 5-HT-- JA receptor peptides according to the present invention which mimic, modulate or inhibit binding of ligands to G-protein coupled receptors.
  • 5-HT-- 1A receptor ligands in the context of the present invention, refer to biological molecules that bind 5-HT-- 1A receptors in vitro, in situ or in vivo, and may include hormones, neurotransmitters, viruses or receptor binding domains, thereof, opsins, rhodopsins, nucleosides, nucleotides, coagulation cascade factors, odorants or pheremones, toxins, colony stimulating factors, platelet activating factors, neuroactive pep ⁇ -ides, neurohumors, or any biologically active compounds, such as drugs or synthetic or naturally occurring compounds.
  • 5-HT-- 1A receptor peptides of greater than 15-25 amino acids are preferred such that the 5-HT--- .A receptor peptides are able to span the lipid bilayer.
  • modified amino acids or chemical derivatives of amino acids of consensus or fragments of 5-HT-- 1A receptors proteins may be provided, which polypeptides contain additional chemical moieties or modified amino acids not normally a part of the protein. Covalent modifications of the peptide are thus i*.'eluded within the scope of the present invention. Such modifications may be introduced into a 5-HT-- 1A receptor peptide by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • 5-HT-- 1A receptor functional domain peptides meeting the above criteria are determined, then such peptides can be synthesized according to knc-vn method steps, wherein solid phase synthesis is preferred (see. e.g., Harlow and
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs may be ob- tained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and/or any subclass thereof.
  • the hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art. See Harlow supra; Ausubel, supra .
  • Chimeric antibodies are molecules having different portions derived from different animal species, or different Ig subclasses of the same or different species, such as those having variable region derived from a murine ⁇ P.b and a human immunoglobulin constant region. Chimeric antibodies and method steps for their production are known in the am (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al. , Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al. , Nature 312:643-646 (1984); Neuberger et al.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immu ⁇ nizing antibody oy producing an antibody to these idiotypic deter ⁇ minants (the anti-Id antibody) .
  • anti-Id antibodies to 5-HT 1A receptors are expected to provide 5-HT-- 1A agonists as described herein, which would this be included as 5-HT-- 1A agonistic antibodies.
  • An anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • mAbs generated against S-100 B may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs.
  • ti-Id mAbs can be coupled to A carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional
  • mice BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a S-100 B epitope.
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an S-100 B epitope, and possessing biological activity of S-100 B .
  • antibody is also meant tr include both in ⁇ tact molecules as well as fragments thereof, sucn as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1982, ) .
  • Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • An "antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • the preferred animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects.
  • treating is intended the administering to subjects of S-100 B , a functional derivative thereof, serotonin, or an agonist or antagonist of the 5-HT, A receptor, for purposes which may include prevention, amelioration, or cure of the diseases discussed below.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, trans- dermal, or buccal routes.
  • Preferred routes for administration of substances which do not cross the blood-brain barrier (such as proteins and larger peptides) to subjects with fully developed blood-brain barriers include intracranial and intracerebroventricular (i.e.v.) routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • 5-HT-- 1A receptor agaonists for engulfonergic or cortical neuron stimulation through induction of S-100 B release
  • 5-HT-- 1A receptor antagonists or anti-S100 B antibodies for up regulation of accumulonergic and/or cortical neurons
  • the antibody is given systemically to the pregnant female or is introduced in utero, for example, into the amniotic cavity.
  • an antibody is administered systemically, for example, by intravenous or intraperitoneal injection.
  • the antibody can cross the blood-brain barrier and enter the brain from the circulation in a young individual in whom the blood-brain barrier is not completely formed, as is well--known to those of skill in the art.
  • the antibody In an individual with a fully formed blood-brain barrier, as in an adult, in order to be effective, the antibody, according to the present invention, must be administered intracranially (i.e.), preferably into the cerebral ventricles (i.e.v.) via a cannula, using methods well-known in the art. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Katzung, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • the present invention contemplates pharmaceutical preparations whirh may also or alternatively contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active com ⁇ pound(s), together with the excipient. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Katzung, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethy...cellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth
  • disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, tal* stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • a number of major developmental disorders involve central serotonergic systems. Autism and Down's Syndrome (DS) are associated with altered serotonergic forebrain innervation, as seen upon postmortem examination of brains from patients with this disorder (Anderson, G.M.
  • Hyperserotoninemia has been observed in autistic children and an antibody specific for the human cortical -HT 1A receptor has been identified in an autistic patient (Anderson, G.M. et al. , supra) .
  • the present invention provides a means for treating an individual affected by autism or DS in early developmental stages, for example, while in utero.
  • S-100 B a functional derivative thereof, or 5-HT 1A agonist is administered to a pregnant woman carrying a fetus diagnosed as ⁇ .ing autism or DS.
  • AD Alzheimer's Disease
  • the present invention provides a method for treating AD by manipulating the serotonergic system to cause release of S- 100 B to stimulate growth of cortical and/or serotonergic neurons.
  • the method, and all other therapeutic methods of the present invention including, but to limited to stimulation of S- 100 B , 5-HT-- 1A receptor stimulation in a subject may have the therapeutic or ciagnostic effect of causing so*'.otonergic neuron growth and/or stimulation, which may be suitable for treatment and/or diagnosis of diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-sleep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers disease, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotoninproduction developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction, is directed to optionally first up-
  • Up-regulation of the 5-HT-- 1A receptors is accomplished by any of a number of means known in the art for depleting central 5-HT-- 1A or blocking its action.
  • Such means include depleting stored serotonin from nerve terminals by agents such as reserpine, fenfluramine or methylene deoxymethamenpetamine (MDMA) (Whitaker- Azmitia, P.C. et al., Eds, Ann. N.Y. Acad. Sci., Vol. 600 (1990)), dietary changes which lower central serotonin evels, such as a tryptophan-deficient diet, or drugs which inhibit 5-HT-- 1A biosynthesis such as parachlorophenylalanine.
  • MDMA methylene deoxymethamenpetamine
  • any treatment which blocks brain corticosteroid levels for example, synthesis inhibitors such as metapyrone or ammoglutethamide
  • corticosteroid action such as corticosteroid antagonists whicn are well-known in the art
  • Treatment with 5-HT--- ⁇ A receptor antagonists including 5-HT-- 1A receptor peptides, or antipeptide antibodies, or anti-Id mAbs such as those described herein, may also achieve the same or similar effects.
  • Such treatment must be performed for a period of time ranging from about 3 days to about 4 weeks prior to stimulation of central serotonergic neurons, as described herein.
  • a period of time ranging from about 3 days to about 4 weeks prior to stimulation of central serotonergic neurons, as described herein.
  • drug or means chosen to up-regulate the receptors, and the age, weight and health of the subject one of ordinary skill in the art will be able to determine the appropriate dose and time course of treatment.
  • the treatment method of the present invention involves providing to the brain a 5-HT-- 1A receptor agonist as described herein, preferably by the oral or parenteral route, in a dose range of about 1 ⁇ g/kg to about 10 mg/kg, for a duration of about 3 days to about 4 weeks, in order to release the S-100 B accumulating in the astroglia.
  • the released S-100 B then acts as a cortical growth factor to stimulate growth of cortical neurons deficient in AD.
  • any pathological process associated with loss of cortical neurons and/or serotonergic neurons or their activity, or lack of normal maintenance of serotonergic innervation can be treated according to the methods of the present invention.
  • neuronal loss may accompany normal aging.
  • the present invention is thus directed to a method of treating neuronal loss in an aging individual with S-100 B , a functional derivative thereof, or a 5-HT 1A agonist which stimulates S-100 B production by astroglial cells in situ. If necessary, as described for AD, prior treatment may be used to up-regulated 5-HT-- 1A receptors in order to allow stimulation of S-100 B release.
  • Affective disorders in particular depression, are diseases with an important serotonergic component.
  • the mode of action of many effective antidepressant drugs is considered to occur via inhibition of 5HT 1A re-uptake, thus prolonging the availability of 5-HT-- 1A to act on post-synaptic 5-HT-- 1A receptors.
  • uptake blockade can occur within minutes of treatment, therapeutic benefits are typically seen only after prolonged (e.g. 3 weeks) of antidepressant therapy (see, for example, Gilman et al. , supra). This difference in time courses suggested to the present inventors that depression involves alterations in levels of serotonergic innervation and synapse formation.
  • stimulation of seroconergic neuronal growth by treatment with S-100 B , a functional derivative thereof, or a 5-HT 1A agonist which stimulates endogenous S-100 B production can be used to treat depression.
  • a subject in need of treatment is administered an effective amount of S-100 B , a functional derivative thereof, or a 5-HT 1A agonist.
  • the treatment with S-100 B , or functional derivatives which do not cross the blood-brain barrier is by i.e.v. infusion.
  • the protein or derivative may be administered systemically.
  • 5-HT 1A agonists, most of which readily cross the blood-brain barrier, are administered systemically.
  • Antidepressant agents have also been useful in the treatment of anxiety and obsessive-compulsive disorders (Gilman et al., supra), indicating the involvement of the serotonergic system in these states.
  • 5-HT-- 1A is involved in synchronization of biological rhythms, and dysregulation may result in sleep disorders (Wauquier, A. et al. , Ann. N.Y. Acad. Sci. 600:447-459 (1990)).
  • serotonin, S-100 B , a functional derivative thereof, or a 5-HT 1A agonist may be used to treat anxiety and sleep disorders due to its action as an inducer of central serotonergic neuronal growth, as described above for treatment of depression.
  • Schizophrenia a major psychiatric illness, is increasingly looked upon as a developmental disorder of the dopaminergic (DA) system wherein central DA activity is increased (Seene, P., Pharmacol. Rev. (1982)).
  • the DA system appears to interact physiologically with serotonergic neurons in a way that stimulation of 5-HT-- 1A results in decrease in DA levels. Therefore, stimulation of growth of serotonergic neurons by administration of S-100 B or a functional derivative thereof, or stimulation of S-100 B release, according to the present invention may be useful in preventing or reversing the effects of enhanced dopaminergic activity, and thus the development of schizophrenia.
  • S-100 B serotonin or precursor thereof or a 5-HT 1A agonist which stimulates astroglial production and/or secretion of S-100 B is administered to a pregnant female carrying a fetus at risk for schizophrenia, either systemically or by intrauterine introduction. Due to its action as a central cortical and/or serotonergic growth factor, S-100 B promotes the growth and maintenance of cortical neurons, most of which utilize glutamate as their neurotransmitter. According to the present invention, S- 100 B , a functional derivative thereof, or a 5-HT 1A receptor agonist may be used to induce repair of cortical neurons following cortical brain damage, such as that associated with traumatic head injury or stroke.
  • Typical dosages comprise about 0.001 to 100 mg/kg body wt such as 0.01-50, 0.05-20, 0.1 to 10 or 1 to 10 mg/kg.
  • the preferred dosages comprise 0.1 to 10 mg/kg body wt.
  • the present invention also contemplates the implantation or transplantation of an astroglial cell capable of producing S-100 B to a subject having a deficit in such cells or having a genetic lesion rendering such cells non-functional, for example, non-responsive to 5-HT ⁇ A receptor stimulation.
  • an astroglial cell capable of producing S-100 B to a subject having a deficit in such cells or having a genetic lesion rendering such cells non-functional, for example, non-responsive to 5-HT ⁇ A receptor stimulation.
  • Such implanted cells may be derived from fetal or adult brain or may be a long term cell line, such as the
  • C6 cell line (Labourdette, G. et al. , supra), which is maintained in culture. Such cells can be implanted in specific target regions in the brain in order to stimulate serotonerg:c cell growth or cortical cell growth, as discussed above.
  • Se-ci also: Azmitia, E.C. et al., Eds., Ann. N.Y. Acad. Sci.., vol.495 (1987)).
  • S-100 B IS A CNS SEROTONERGIC GROWTH FACTOR METHODS
  • cells were plated on 8-well glass culture chamber slides (Miles Scientific) i.:. r 30h, fixed with ice-cold 4% paraformaldehyde and reacted with a specific 5-HT antibody (1/4000 dilution; Incstar, Stillwater, MI) and an avidin- biotin secondary (Vector Labs) .
  • the 5-HT immunoreactive neurons were analyzed using a Bioquant computer imaging system.
  • the slides were coded and all measures taken blind by a naive observer. On each well, 10 areas (0.5 mm 2 ) were randomly selected and the largest 5-HT-IR neuron in the field measured for somal area and neurite length (Azmitia, E.C, et al., 1987, supra) .
  • S-100 B was added at initial plating for 30h to mesencephalic cultures and the 5-HT neurons were immunocytochemically stained.
  • DISCUSSION The results indicate that S-100 B is an SGF.
  • EGF, insulin and calmodulin were not found to produce any stimulation, indicating that the SGF activity of S-100 B was not a byproduct of its Ca 2+ binding potential nor was it due to a general mitogenic potential.
  • S-100 in the brain is an astroglial specific protein (Isobe, T. et al. , supra) . Recent results have shown that the SGF properties of 5-HT JA -stimulated, glial cell conditioned medium is blocked by treatment with an anti-S-100 antibody (see Example II, below) .
  • astroglial cells were stimulated in primary culture with 100 nM ipsaperone (IPS) , a 5-HT 1A receptor agonist, and collected the conditioned media (GCM-IPS) . vie then added the GCM-IPS to primary cultures of serotonin neurons, with and without the addition of an antibody to S-100, and assessed the effects on neuronal growth.
  • IPS ipsaperone
  • GCM-IPS conditioned media
  • the cells were left to incubate at 37°C for twelve hours before replacing media with fresh serum-free media containing 100 nM ipsapirone, a selective 5-HT 1A receptor agonist. After 24 hr, the media (referred to as GCM-IPS) was collected and stored at -70°C until tested in neuronal cultures.
  • GCM-IPS The growth-promoting properties of native bovine S-100
  • the S-100 was obtained from East Acres Biologicals, Southbridge,
  • S-100 or GCM-IPS or each of thes.e in the presence of a polyclonal antibody to S-100 (Accurate Chemical, Westbury, NY; final dilution 1/10,000) were added at the time of neuronal plating.
  • the polyclonal antibody had been characterized in our laboratory and shown to be positive for immunocytochemical staining of astrocytes in culture and brain) .
  • astroglial cultures After exposure to serum-free media with or without 100 nM ipsapirone, cultures were rinsed twice with Tris-buffered saline (TBS) at 4°C before incubation with a polyclonal antibody to a specific astroglial marker, glial fibrillary acidic protein (GFAP) (Ac-curate Chemicals; final dilution 1/800 in TBS with 0.2% Triton and 0.1% normal swine serum) for 2 hrs at 37°C After rinsing with TBS, the cultures were stained using the avidin/biotin method prepared as Vectastain (Vector Labs) with final visualization using diaminobenzidine.
  • TBS Tris-buffered saline
  • GFAP glial fibrillary acidic protein
  • the morphological alterations were characterized by an increase in process-bearing cells and an increased colonization of the cells. These changes were consistently observed in all eight primary cultures (ie. from eight different litters of animals) used to derive GCM.
  • DISCUSSION It had been previously demonstrated that serotonergic neurons regulate their own growth through activation of a 5-HT j receptor (Whitaker-Azmitia, P.M. et al. , Neurosci. Lett. 67:307-312 (1986) ) .
  • the present inventors found that astroglial cells contain high levels of -T-HTj receptors in the immature state (Whitaker- Azmitia, P.M. et al. , J. Neurochem.
  • 5-HT jA receptor activation of a subtype of these receptors, the 5-HT jA receptor, leads to secretion into the medium of a factor which can stimulate serotonergic maturation in dissociated tissue culture preparation (Whitaker-Azmitia, P.M. et al. , Brain Res. 497:80-85 (1989)) .
  • 5-HT 1A receptors on brain astroglial cells appear to be involved in the release of S-100. Therefore, S-100 provides at least one means by which serotonin can autoregulate development of serotonergic nerves .
  • NGF nerve growth factor
  • astroglial 5-HT receptor Stimulation of astroglial 5-HT receptor causes astroglial cells to acquire a more mature morphology and to release a factor (or factors) which promotes growth of serotonergic neurons.
  • a factor or factors which promotes growth of serotonergic neurons.
  • S-100 the astroglial-specific protein S-100. This may be a particularly important observation, in view of studies implicating S-100 in both Down's Syndrome and Alzheimer's Disease, as discussed above.
  • EXAMPLE III ANTIPEPTIDE ANTIBODIES AGAINST THE 5-HT 1A RECEPTOR
  • a method for selecting two new sites for anti-5-HT-- 1A receptor antibody recognicion against the 5-HT 1A receptor S1A-170 (aa 170- 186) and S1A-258 (aa 258-274) .
  • S1A-170 aa 170- 186
  • S1A-258 aa 258-274
  • S1A-170 aa 170- 186
  • S1A-258 aa 258-274
  • the receptor is homologous to the beta-adrenergic receptor family and many of the various segments of the 5-HT 1A receptor can be inferred from the extensive work with' the ⁇ -adrenergic receptor.
  • the agonist binding site consists of a least two aspartate (asp) residues within the 2nd and 3rd transmembrane regions (Dohlman et al.
  • peptide sequences can be selected indicate the anatomical location of various segments of the full molecule and deterr.iine the cellular distribution of particular functional regions.
  • the antigenic sites on a peptide can be approximated based on the hydrophilicity score which assumes that the greater the local hydrophilicity, the more antigenic the sequence (Hopp, T.P. Proc. Natl. Acad. Sci. USA 8:3824-3828 (1981)) .
  • This measure assigns a numerical value to the various amino-acids; for example K, R, D and ⁇ l have a value of +3.00, W has a value of -3.4, and G and P have a value of 0. the calculated window average at a residue is calculated across 6 residues.
  • the hydrophilicity score for A1A170 is shown in Table One.
  • the net charge of a peptide sequence should be near neutrality. If the molecule is too highly charged it will present problems durinj the purification procedure after the peptide is synthesized. If the net charge is highly basic or acidic a cation or anion exchange resin can be used. Bio-rad AG-50 resin has been successfully used for very basic peptides. However, strong deviations from neutrality is also a problem during the attachment to KLB which should proceed at neutral pH (see below) . S1A-170 has 6 charged residues and a net - 2 charge while S1A-258 has 5 charged residues and a net + 1 charge. (5) Amino Acid length. The sequence for an ideal peptide for antibody formation should have 15-20 amino acids.
  • a strand of 6 amino acids is the lower limit for a recognition site whole more than 20 presents some additional problems with the synthesis and structural considerations (Harlow, E. et al. Antibodies: A Laboratory Manual Cold Spring Harbor Press (1988)) . Both S1A-170 and S1A-258 have 17 residues.
  • a protein molecule has many possible phosphorylation and glycosylation sites. These sites should be avoided in choosing a sequence unless a particular confirmation is sought. Antibodies have been raised against phosphorylated sequences but these antibodies have altered affinity for the un-phosphorylated site (See Czernik et al. Method Enzymol 201:264-283 (1991). Furthermore, a phosphorylated segment of the molecule often confers allosteric changes in the protein structure which may reduce the affinity for the peptide segment artificially produced. It can be appreciated, that sites adjacent to modified sites may be less desirable for the same reasons.
  • the glycosylation sites are located on the Asparagine (ASN, N) residues at positions 10, 11 and 24.
  • Cysteine (Cys, C) residues are commonly involved in disulfide bridges. For chis reason it is advisable to avoid a Cys residue in the middle of a peptide sequence.
  • the side chain-protected amino acids were: Arg (Mts) ; His (BOM); Thr (Bzl) ; Cys (4-CH3-Bzl); Trp (CHO); Ser (Bzl; Glu (OBzl) ; and Asp (cHex) . Double coupling was necessary for several amino acids such as Trp, Leu, Thr, Glu, Ser, Cys and Val.
  • the peptides were deprotected and cleaved from the resin with liquid FH at -10°C for 2 h in the presence of 5% (v/v) anisole and 5% (v/v) dimethyl sulfide.
  • the peptide was precipitated with ethyl either, and solubilized in 6M Guanidine HC1 @ 10 mg/ml (41 ml) .
  • the formyl group was removed from the indole portion of Trp bv treatment with HF.
  • the sample was cooled to 0°C in a salt ice bath in a round bottom flas ⁇ with stirring.
  • Ethanolamine was added at a final concentration of IM (2.5 ml) and stirred for 4 hr. at 0°C The temperature was critical with the pH > 8 since a low temperature prevents the cyclization of glutamic acid and aspartic acid. The reaction was quenched by reducing the pH ⁇ 7.0 with [HCL] (Baker, HPLC grade). The sample was filtered
  • the peptides, CSH 228 and 229 were coupled to keyhole limpet hemocyanin (Sigma) via maleimidobenzoyl-N-hyroxysuccinimide
  • KLH Keyhole limpet hemocyanin
  • MBS MBS
  • Pierce N22312
  • dimethylformamide Burdick and Jackson, Baxter, N.J.
  • the MBS solution was very slowly added to the KLH solution, one drop at a time while stirring and the mixture allowed to mix for 30 min at RT.
  • the unbound MBS was removed by filtration on Sephadex G25 in PBS (Pharmacia) .
  • the peptides were dissolved in PBS at 10 mg/ml and added slowly to the
  • the blood was kept at 5°C and spun once at low speed for 10 min and the serum spun a second time in Eppendorf tubes for 10 min before being aliquoted and frozen.
  • Serum antibody titer was determined by radioimmunoassay. Wells of 96-well polyvinylchloride microtiter plates (Falcon Microtest III) were coated with 50 ⁇ l of the appropriate peptide (1 mg/10 ml) for at least 3 hr at RT. The plates were washed 3 times with PBS and unbound sites were saturated with POO ⁇ l of 3% (w/v) bovine serum albumin (BSA) . Dilutions of immune and preimmune serum were added to wells at concentration from 1/50 to 1/50,000 for at least lh at RT.
  • BSA bovine serum albumin
  • Elec rophoresis was performed on 1mm 12.5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulfate using the buffer system of Laemmli (1977) .
  • 20 and 40 ug of tissue from hippocampus was run in each well along with 5ul of standard (Bio Rad biotinylater SDS-PAGE standards, low range•cat # 161-0306).
  • the hippocampus was removed from a young Long Evans female rat (100-150 gm; Charles River, Comments, NY) and immediately frozen in liquid nitrogen. A single hippocampus was transferred to lysis buffer (see below) containing 2% SDS.
  • the tissue was immediately homogenized by hand in a 1.5 ml polypropylene eppendorf tube (pellet pestle with disposable tube, #74920, Kontes, N.J.) and allowed to sit for 15 min on ice before spinning in a microfuge Eppendorf Microcentrifuge 5414) at 5°C for 15 min. The supernatant was collected and assayed for protein amount using a commercial dye-binding microassay (Bio Rad) . Routinely, 3ul were added to 1ml of solution and lead on a spectrophotometer at wave length 595 nm. The solution was adjusted to a final concentration of 2 mg/ml.
  • the supernatant was mixed with an equal amount of sample buffer (Laemmli) , ⁇ -mercaptoethanol added to a final concentration of 10% (V/v) and the sample boiled for 5 min.
  • sample buffer Laemmli
  • ⁇ -mercaptoethanol added to a final concentration of 10% (V/v) and the sample boiled for 5 min.
  • the gel •• •/as run for 5 hr. at 100 volts on a vertical gel electrophoresis apparatus. Proteins were transferred electrophoretically to nitrocellulose using a Bio- Rad Trans Blot Cell overnight at 50 volts in the cold room (Towbin et al. , 1979 & 1984) .
  • the nitrocellulose was incubated with antiserum at dilutions from 1/100 to 1/10,000 in 0.1% (v/v) Tv.*een-20 (Sigma) in Tris buffered (O.lM, pH 7.4) saline (0.9%) solution (TTBS) . It is our experience that the best results were obtained with our antibodies at dilution of 1/1000 to 1/5000. The antibody at lower dilutions (1/250) gave increase background and less sensitivity.
  • the avidin-biotin peroxidase procedure was used to identify the protein band as described by Vector Laboratories. Briefly, the nitrocellulose sheets were cut to include a iitandard and the appropriate rows and washed in small 75mm disposable Petri dishes.
  • nitrocellulose strips were incubated with the antisera for at least 2 hr at 40°C (the strips would be left with antisera for several days at RT or in the Cold room) .
  • the strips were rinsed three times in TTBS for a total of 10 min on a shaker between biotinylated secondary (30 min incubation) , the ABC reagents (20 min incubation) , and the DAB peroxide reaction.
  • the strips were first incubated with freshly filtered diaminoben ⁇ ;idine (5 mg/20 ml TBS; Sigma) and 0.2% Nickel ammonium sulfate for 5 min at RT and the H 2 0 2 added at a final concentration of 0.01% (v/v) .
  • Boehringer- Mannheim produces biotinylated reagents that are easier and cheaper to use than Vector Stains products but we have not yet compared their sensitivity.
  • Lysis Buffer (Draeta et al. Cell 54:17-26 (1988)) was made from highest grade chemicals from Sigma (St. Louis) and Boehringer-Mannheim: 50mM Tris pH 7.4; 150 mM NaCl; 1% NP-40; lOmM EDTA; ImM MgCl 2 ; 1 mM CaCl 2 ; 10% Glycerol; 400 ⁇ M Sodium Orthovanadate; 50 mM NaFluoride; 50mg/l PMSG (phenylmethane sulfonylfluoride) from lOmg/ml isopropanol; 1 mg/leupeptin; 10 mg/1 Soybean Trypsin Inhibitor; lmg/1 Aprotinin and 10 mg/1 of TPCK (L- l-chiloro-3- [4-tosylamido] -4]phenyl-2-butanone) from 3 mg/ml of ethanol. Immun
  • Neonatal (1-2 weeks) and adult female rats were perfused with a variety of fixat.* /es and prepared for immunocytocliamistry according to our published procedures (Azmitia et al. J. Neuroscience 3:2083- 2090 (1983) .
  • Rats Sprague-Dawley, Female, Taconic Breeders, 220 gm
  • monkeys Macaca Fascicularis, female, Charles River Breeding Laboratory, 3.3 kg
  • the glutaraldehyde and acrolein fixatives were continued after 10 min with the same solution with only paraformaldehyde (total perfusion volume was 100 ml for neonate, 250 ml for the adult rats and 1500 ml in the monkey) for an additional 20 min.
  • the brains were profixed at 5°C for at least 4 hr before being processed for immunocytochemistry. Thirty-micrometer sections of the hippocampus and brainstem were cut on a Vibratome (Oxford) .
  • the primary anti-serum and the se >ndary sera were diluted in 0.1 M Tris buffered (pH 7.4) saline (0.85%) containing 1% normal sheep serum and 0.1% Triton X-100.
  • the sections were incubated for 18-72 hr at 5°C followed by 2 hr at room temperature (RT) in antipeptide antibody serum at a dilution of 1/1000- 1/10,000.
  • the sections were then processed with the elite Vector stain ABC-kit as directed by the manufacturer.
  • Specificity of the antibody raised against synthetic peptides is increased compared to using the ful. " molecule since a specific region of the structure is targeted. Regions with high homologies with other molecules can be avoided. A functional region (3rd cytoplasmic loop) or a structural portion (second extracellular loop) can be selected for study (Fig. 6) .
  • the antibody peptide can be produced as soon as the primary cDNA structure has been demonstrated.
  • High titers with the antipeptide antibody can be obtained because the peptide is bound to a carrier protein that provides highly immunogenic sites for T-cell receptor binding.
  • a carrier protein that provides highly immunogenic sites for T-cell receptor binding.
  • Anatomical and cellular localization of the receptor can be performed in neonatal and adult rats as well as adult monkey. All fixation solutions produced good results.
  • the best staining of CNS was in neonates and this confirms the ligand binding studies which have shown higher values for the 5-HT 1A receptor during early prenatal periods (Bar-Peled et al. Neurosci. Lett.
  • Characterization of the antipeptide antibody must establish that the native protein is selectively recognized, such that the following considerations are preferably addressed.
  • 8-OH-DPAT for . s ance does not recognize the receptor if it is not linked to a G-protein.
  • the antibody on the other hand, can recognize all the receptor molecules even those in transit from the cell body to the dendritic plasma membrane via microtubules (see
  • Immunostaining of a single band in Western analysis is usually considered an indication of the general specificity of an antibody.
  • this requires careful manipulation of the antibody dilution based and preparation of t.-.e tissue sample. Protein fragments or aggregation of the molecvle can result in several bands on a Western even if the antibody only recognizes a single protein. For this reason, we use a special lysis buffer and treat the tissue with reducing and denaturing conditions (such as B-mercaptoethanol and boiling) .
  • the antibody JWR21 (242-267) was shown to precipitate the [ 125 I]N 3 -NAPS photoaffinity labelled receptor (Raymond et al. Molecular Pharmacology 36:015-021 (1989)).
  • the 5-HT 1A receptor antibody against 243-268 precipitated the binding sites of 3 lH-8-OH-DPAT when protein A- sepharose CL-4B was added. No influence of the antiserum alone was seen in the binding.
  • the synthesis was performed with a 4J-JA ABI sequencer.
  • the alternative is TFMSA cleavage which can be done at the bench.

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Abstract

Un procédé permet de stimuler chez un sujet la production de S-100B en lui administrant une quantité efficace d'un agoniste d'un récepteur de 5-HT--1A, l'agoniste pouvant être le récepteur ou un anticorps antipeptide d'un domaine fonctionnel d'un récepteur de 5-HT--1A. On décrit des procédés complémentaires permettant de stimuler la croissance des neurones centraux sérotoninergiques en les mettant en contact avec une quantité efficace de S-100B, ou d'un de ses analogues ou dérivés, et la croissance de ces neurones centraux sérotoninergiques est inhibée si on les met en contact avec une quantité efficace d'un inhibiteur de la production ou de l'action de S-100B. De plus, ces procédés permettent de traiter des maladies, liées à une innervation ou activité sérotoninergique centrale amoindrie, qui incluent l'autisme, la dépression, l'anxiété, des troubles du sommeil basés sur les rythmes biologiques et des lésions du cortex. On peut traiter des maladies liées à une innervation sérotoninergique centrale accrue mais à une libération astrogliale inefficace de S-100B, telles que la trisomie 21 et la maladie d'Alzheimer, par la régulation positive des récepteurs astrogliaux de 5-HT--1A suivie par une stimulation de production ou de libération de S-100B en utilisant un agoniste d'un récepteur de 5-HT--1A, y compris un anticorps antipeptide d'un domaine fonctionnel d'un récepteur de 5-HT--1A.
PCT/US1993/010095 1992-10-23 1993-10-22 Interactions fonctionnelles entre le s-100b glial et les neurones serotoninergiques du systeme nerveux central WO1994009765A1 (fr)

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WO2002060423A3 (fr) * 2001-01-29 2003-04-10 Otsuka Pharma Co Ltd Agoniste du sous-type du recepteur 5-ht¿1a?
US7053092B2 (en) 2001-01-29 2006-05-30 Otsuka Pharmaceutical Co., Ltd. 5-HT1a receptor subtype agonist
US7910589B2 (en) 2001-09-25 2011-03-22 Otsuka Pharmaceutical Co., Ltd. Low hygroscopic aripiprazole drug substance and processes for the preparation thereof
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US5254552A (en) * 1988-05-24 1993-10-19 American Home Products Corporation Aryl-and heteroaryl piperazinyl carboxamides having central nervous system activity

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US5254552A (en) * 1988-05-24 1993-10-19 American Home Products Corporation Aryl-and heteroaryl piperazinyl carboxamides having central nervous system activity
US5070102A (en) * 1988-09-20 1991-12-03 Troponwerke Gmbh & Co. Medicaments for the treatment of cerebral apoplexy

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PSYCHOPATHOLOGY, Volume 22, Supplement 1, issued April 1989, M.S. EISON, "The New Generation of Serotonergic Anxiolytics: Possible Clinical Roles", pages 13-20. *

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US9387207B2 (en) 2001-01-29 2016-07-12 Otsuka Pharmaceutical Co., Ltd. 5-HT1A receptor subtype agonist
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