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WO1999020273A1 - Traitement de la schizophrenie par utilisation d'un inhibiteur de la dephosphorylation de la darpp-32 - Google Patents

Traitement de la schizophrenie par utilisation d'un inhibiteur de la dephosphorylation de la darpp-32 Download PDF

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Publication number
WO1999020273A1
WO1999020273A1 PCT/US1998/022127 US9822127W WO9920273A1 WO 1999020273 A1 WO1999020273 A1 WO 1999020273A1 US 9822127 W US9822127 W US 9822127W WO 9920273 A1 WO9920273 A1 WO 9920273A1
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WIPO (PCT)
Prior art keywords
darpp
phosphorylation
agent
dopamine
calcineurin
Prior art date
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PCT/US1998/022127
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English (en)
Inventor
Akinori Nishi
Gretchen L. Snyder
Allen A. Fienberg
Paul Greengard
Original Assignee
The Rockefeller University
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Filing date
Publication date
Priority claimed from US08/953,442 external-priority patent/US6013621A/en
Application filed by The Rockefeller University filed Critical The Rockefeller University
Priority to AU11036/99A priority Critical patent/AU1103699A/en
Publication of WO1999020273A1 publication Critical patent/WO1999020273A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • A61K31/33—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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/302—Schizophrenia

Definitions

  • the present invention provides a method for treating schizophrenia and related disorders.
  • the present invention provides methods for identifying agents that can be used in the treatment of schizophrenia and related disorders.
  • DARPP-32 a dopamine- and cyclic AMP (cAMP)-regulated phosphoprotein having a molecular weight of 32 kilodaltons
  • cAMP dopamine- and cyclic AMP
  • the human DARP-32 has been sequenced [Brene et al, J. Neuroscience, 14:985-998 (1994)].
  • DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on a single threonine residue, thr 34 , resulting in its conversion into a potent inhibitor of protein phosphatase-1 (Hemmings et al, 1984). DARPP-32 can be dephosphorylated and inactivated in vitro by the calcium/calmodulin-dependent protein phosphatase, calcineurin (King et al., 1984).
  • PKA cAMP-dependent protein kinase
  • Dopamine has been shown to stimulate the phosphorylation of DARPP-32 in neostriatum by activation of a biochemical cascade involving stimulation of Dl receptors, activation of adenylyl cyclase, increased cAMP formation and increased activity of PKA (Walaas and Greengard, 1984).
  • the selective enrichment of DARPP-32 in dopaminoceptive neurons and its regulation by dopamine strongly indicate that DARPP-32, by regulating protein phosphatase- 1 activity, plays a key role in mediating the effects of dopamine on these cells.
  • the control of protein phosphatase- 1 activity by DARPP-32 is likely to have a significant role in the regulation of neuronal excitability.
  • Dl and D5 subtypes D1 and D2 subfamily
  • D2 subfamily D2, D3 and D4 subtypes
  • Dl and D2 dopamine receptors are abundantly expressed on cell bodies and dendritic processes of medium spiny neurons (Levey et al, 1993).
  • RNAs coding for each of the other dopamine receptor subtypes have been isolated from individual neostriatal neurons (Surmeier et al., 1996), but whether these receptor proteins are expressed in medium spiny neurons and how they functionally interact with Dl and D2 receptors is still unclear.
  • Dl and D2 receptors have opposing actions on the activity of adenylyl cyclase in neostriatal neurons; whereas activation of Dl receptors increases cAMP formation by adenylyl cyclase, D2 receptors inhibit adenylyl cyclase activity (Stoof and Kebabian, 1981).
  • D2-like dopamine receptors via interactions with specific G-proteins can be coupled to multiple effector systems, including calcium channels, potassium channels and phospholipase C (for review, see Huff, 1996).
  • effector systems including calcium channels, potassium channels and phospholipase C
  • Yan et al, (1996) have shown that D2 receptors on neostriatal neurons negatively couple to calcium channels through a G I/0 class protein.
  • activation of D2 receptors apparently decreases sodium currents in medium spiny neostriatal neurons through a membrane-delimited pathway and increases these currents through a soluble second messenger pathway (presumably involving inhibition of adenylyl cyclase) (Surmeier et al, 1992).
  • Schizophrenia is a clinical ailment which has an immense effect on the public health. For example, it has been estimated that as many as 50% of the homeless people living in the United States are schizophrenic. [Bachrach, In: Treating the Homeless Mentally III, Washington, D.C., American Psychiatric Press, 13-40, Lamb et al. ed. (1992)]. In addition, approximately 2.5%) of the total dollars spent for health care in the United States is spent in the treatment of schizophrenia [Rupp et al, Psychiatric Clin. North Am. 16:413-423 (1993)]. Unfortunately, at present, there exists no reliable or effective means of treating schizophrenia. Therefore, there is a need to provide new drugs assays which can be used to develop new drugs to treat schizophrenia. Further there is a need to provide new drugs which can be used in such treatment.
  • the present invention provides a novel means of exploiting the determination that the phosphorylation of DARPP-32 is under bidirectional regulation involving dopamine, dopamine Dl and D2 receptors, and calcineurin.
  • the present invention further provides new methodology in the treatment of diseases such as schizophrenia, Parkinson's disease, Tourette's syndrome, drug abuse and attention deficit disorder.
  • the present invention provides methods for identifying agents that can be used in such treatment.
  • the present invention provides a method of treating a schizophrenic patient that comprises administering an agent to the patient that inhibits the dephosphorylation of thr 3 -phosphorylated DARPP-32.
  • an agent to the patient that inhibits the dephosphorylation of thr 3 -phosphorylated DARPP-32.
  • the agent inhibits calcineurin.
  • the agent inhibits calcineurin by binding to calcineurin.
  • the agent readily passes through the blood brain barrier.
  • One such agent is FK506 [Liu, Trends in Pharm. Sci., 14:182-188 (1993)].
  • the agent can be modified or otherwise altered so that it can cross or be carried across the blood brain barrier.
  • the present invention also provides a method of identifying an agent for use in the treatment of a schizophrenic patient.
  • the agent is selected for its ability to inhibit the dephosphorylation of thr 34 -phosphorylated DARPP-32 by calcineurin.
  • One such embodiment comprises using calcineurin or a fragment or analog thereof in a drug screen.
  • calcineurin is bound to a solid support.
  • Compounds that bind to calcineurin or the fragment or analog thereof are selected as potential agents.
  • a potential agent is contacted with calcineurin and a phosphorylated substrate for calcineurin.
  • the amount of dephosphorylation of the substrate is determined and a potential agent is identified if a decrease in the dephosphorylation of substrate is determined in the presence of the potential agent.
  • the phosphorylated substrate is thr 34 -phosphorylated DARPP-32.
  • a potential agent is administered to an animal along with a dopamine D2 receptor agonist.
  • the administration of the D2 agonist alone results in a diminished percentage of prepulse inhibition.
  • the response of the mouse to prepulse inhibition is measured and an agent is selected when the response to prepulse inhibition is statistically significantly increased in the presence of the potential agent relative to that determined in the absence of the potential agent.
  • the selected agent is also administered to a DARPP-32 knockout mouse.
  • a DARPP-32 knockout mouse has been shown to have a diminished percent prepulse inhibition relative to the wildtype mouse, see below.
  • the response of the knockout mouse to prepulse inhibition is then determined. An agent is identified when the response to prepulse inhibition is not statistically significantly increased in the DARPP-32 knockout mouse in the presence of the selected agent relative to that determined in the absence of the selected agent.
  • the animal is a mammal.
  • the animal is a rodent.
  • the rodent is a mouse.
  • any and/or all of the above embodiments for identifying an agent for use in the treatment of a schizophrenic patient can be combined to form additional drug screens and assays, all of which are contemplated by the present invention.
  • Figure 1 depicts the effect of dopamine on the level of phosphorylated DARPP-32 in neostriatum. Slices were incubated with dopamine (100 ⁇ M) in the presence of the dopamine uptake inhibitor nomifensine (10 ⁇ M) for the indicated times.
  • the phosphorylated DARPP-32 was detected at a molecular mass of approximately 32 kDa using a monoclonal antibody (Ab), mAb-23, against thr 34 -phosphorylated DARPP-32.
  • the phospho-DARPP-32 antibody also detected a cross-reactive protein band at a molecular mass of approximately 75 kDa, the levels of which were not affected by dopamine.
  • FIG. 2 Effects of a Dl agonist, SKF82526, and a D2 agonist, quinpirole, on the basal level of phosphorylated DARPP-32 in neostriatum.
  • Figure 2A slices were incubated with 1 ⁇ M SKF82526, whereas in Figure 2B slices were incubated with 1 ⁇ M quinpirole for the indicated times.
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with untreated tissue. Data represent means ⁇ SEM for 4 to 12 experiments.
  • Figure 3 depicts the opposing effects of a Dl agonist, SKF82526, and a D2 agonist, quinpirole, on the level of phosphorylated DARPP-32 in neostriatum.
  • Slices were preincubated with the indicated concentrations of quinpirole (1 nM to 1 ⁇ M) for 5 minutes and then incubated with quinpirole /J/MS 1 ⁇ M SKF82526 for an additional 5 minutes.
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent means ⁇ SEM for 4 to 5 experiments. * P ⁇ 0.01 compared with SKF82526 alone.
  • Figure 4 depicts the effect of the antipsychotic drug raclopride on the level of phosphorylated DARPP-32 in neostriatum ( Figure 4A) and nucleus accumbens ( Figure 4B). Slices were incubated for a total of 20 minutes. Raclopride (1 ⁇ M) was added at 0 min, quinpirole (100 nM) at 10 min and SKF82526 (1 ⁇ M) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. In Figure 4A the data represent means ⁇ SEM for 6 to 9 experiments.
  • FIG. 5 Effect of a D2 agonist, quinpirole, on stimulated levels of phosphorylated DARPP-32 in neostriatum.
  • Slices were preincubated with quinpirole (1 ⁇ M) for 5 min and then incubated with quinpirole plus either 1 ⁇ M SKF82526, in Figure 5 A, 10 ⁇ M forskolin in Figure 5B, or 1 mM 8-bromo-cAMP in Figure 5C for an additional 5 minutes.
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin or 8-bromo-c AMP alone. Data represent means ⁇ SEM for 3 to 4 experiments. * P ⁇ 0.01 compared with SKF82526, forskolin or 8-bromo-cAMP alone.
  • Figure 6 depicts the effect of Ca 2+ -free/EGTA medium on the level of phosphorylated DARPP-32 in neostriatum. Slices were incubated in control or Ca 2+ -free/EGTA medium for 20 minutes.
  • phosphorylated DARPP-32 was detected using a phosphorylation-state specific antibody (Ab), mAb-23.
  • Ab phosphorylation-state specific antibody
  • Figure 6B the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control. Data represent means ⁇ SEM for 4 experiments. * P ⁇ 0.01 compared with control.
  • Figure 7 depicts the absence of effect of a D2 agonist, quinpirole, on the level of phosphorylated DARPP-32 in Ca + -f ⁇ ee/EGTA medium.
  • Neostriatal slices were incubated in Ca 2+ -free/EGTA medium for a total of 20 minutes. Buffer was replaced by Ca 2+ -free/EGTA medium at 0 minutes, quinpirole (1 ⁇ M) was added at 10 min and SKF82526 (1 niM), in Figure 7A, forskolin (10 ⁇ M) in Figure 7B, or 8-bromo-cAMP (1 mM) in Figure 7C, at 15 minutes of incubation.
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin or 8-bromo-cAMP alone. Data represent means ⁇ SEM for 3 to 4 experiments.
  • DARPP-32 Activation of dopamine D2 receptors decreases DARPP-32 phosphorylation by two mechanisms (which might occur in the same or in different groups of neurons): one involves an inhibition of adenylyl cyclase, a decrease in cAMP, a decrease in activity of PKA and a decreased phosphorylation of DARPP-32; the other involves an increase in intracellular Ca 2+ , an activation of calcineurin and an increased dephosphorylation of thr 34 -phospho-DARPP-32.
  • FK506 increases the level of DARPP-32 phosphorylation in neostriatal slices. Rat neostriatal slices were incubated in the absence (control) or presence of forskolin (10 ⁇ M) alone; or forskolin and FK506 using the various concentrations shown. The results demonstrate that inhibition of calcineurin activity by FK506 increases DARPP-32 phosphorylation in a dose-dependent manner.
  • Figure 10 depicts a plot of the ratio of DARPP-32 levels (in the dorsilateral prefrontal cortex) of post-mortem samples from brains of schizophrenics divided by the matched control brains. Pair numbers refer to the particular schizophrenic patient compared to its matched non-schizophrenic control. Levels of DARPP-32 were determined by immunoblotting.
  • Figure 11 depicts a Plot of % Prepulse Inhibition versus Prepulse Intensity for wild type and DARPP-32 Mutant Mice. An acoustic startle paradigm was used as described by Wan et al. [Psychopharmacology, 113:103-109 (1993)].
  • DARPP-32 phosphorylation is regulated in mouse neostriatum through the opposing actions of Dl -like and D2-like dopamine receptors.
  • Previous reports [Walaas et al, (1983); Walaas and Greengard, (1984)] had shown that dopamine stimulates Dl-like dopamine receptors in rat striatum, leading to sequential activation of adenylyl cyclase and cAMP-dependent protein kinase; and phosphorylation of DARPP-32 on threonine-34 (thr 34 ).
  • D2 receptors on DARPP-32 phosphorylation is calcium-dependent and mediated by an increase in intracellular Ca 2+ and an activation of calcineurin.
  • the present invention is consistent with the widely believed premise that a relative hyperactivity within mesolimbic and/or nigrostriatal dopaminergic systems contribute to the etiology of schizophrenia (Davis et al, 1991). This premise is based largely on studies of the mechanism of action of neuroleptic medications. Thus the therapeutic efficacy of antipsychotic drugs is linked to their ability to block dopamine receptors, particularly those which interact with D2-like dopamine receptors (Seeman, 1992).
  • Raclopride also blocked D2-mediated inhibition of D 1 -stimulated DARPP-32 phosphorylation in neostriatum and nucleus accumbens.
  • the nucleus accumbens is a target for mesolimbic dopaminergic projections (Swanson, 1982) that has been implicated in the genesis of psychotic symptoms (Davis et al, 1991).
  • Dl antagonists like D2 agonists, would be expected to inhibit increases in DARPP-32 phosphorylation.
  • the Dl/DARPP-32/protein phosphatase- 1 cascade regulates the state of phosphorylation and/or the activity of, the electrogenic sodium pump, Na + ,K + -ATPase (Nishi et al, 1996), calcium channels (Surmeier et al, 1994), voltage-dependent sodium channels [Surmeier et al, (1992); Schiffman et al, (1994)] and the glutamate receptor subunit, NRl (Snyder et ⁇ /., 1996).
  • present invention provides novel methodology for the treatment of diseases such as schizophrenia, Parkinson's disease, Tourette's syndrome, drug abuse and attention deficit disorder through the use of drugs that interfere with the calcineurin-dependent dephosphorylation of DARPP-32. Furthermore, the present invention provides methods for selecting and/or screening for drugs that can be used in treatment of these diseases.
  • a potential inhibitor of calcineurin could be obtained by screening a random peptide library produced by recombinant bacteriophage for example, [Scott and Smith, Science, 249:386-390 (1990); Cwirla et al, Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al, Science, 249:404-406 (1990)] or a chemical library. Using the "phage method” very large libraries can be constructed (10 6 -10 8 chemical entities).
  • a second approach uses primarily chemical methods, of which the Geysen method [Geysen et al, Molecular Immunology 23:709-715 (1986); Geysen et al. J.
  • chemical analogues can be either selected from a library of chemicals as are commercially available from most large chemical companies including Merck, Glaxo Welcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, or alternatively synthesized de novo.
  • the prospective agent can be placed into any standard assay to test its effect on the dephosphorylation of DARPP-32 by calcineurin.
  • a drug is selected that inhibits the dephosphorylation.
  • the present invention contemplates screens for small molecules, analogs thereof, as well as screens for natural inhibitors that bind to and inhibit calcineurin in vivo.
  • natural products libraries can be screened using assays of the invention for molecules that antagonize calcineurin activity.
  • the agent can cross the blood-brain barrier, which would allow for intravenous or oral administration.
  • Many strategies are available for crossing the blood-brain barrier, including but not limited to, increasing the hydrophobic nature of a molecule; introducing the molecule as a conjugate to a carrier, such as transferrin, targeted to a receptor in the blood-brain barrier; and the like.
  • the molecule can be administered intracranially or, more preferably, intraventricularly.
  • osmotic disruption of the blood-brain barrier can be used to effect delivery of agent to the brain ([Neuwelt] et al, 1995, Proc. Natl. Acad. Sci.
  • an agent can be administered in a liposome targeted to the blood-brain barrier.
  • Administration of pharmaceutical agents in liposomes is known ⁇ see Langer, 1990, Science 249:1527-1533; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) All of such methods are envisioned in the present invention.
  • the octanol-water partition system only provides a qualitative indication of the capability of a compound to cross the blood-brain barrier.
  • comparisons between known histamine H 2 receptor antagonists suggest that there is no such simple relationship between their brain penetration and octanol water partition coefficients (Young et al, 1988, J. Med. Chem. 31:656).
  • Other factors, besides the octanol- water partition influence the propensity to cross the blood-brain barrier.
  • Comparison of the ability of histamine H 2 receptor antagonists to cross the blood-brain barrier suggests that brain penetration may increase with decreasing over-all hydrogen binding ability of a compound (Young et al, supra). Begley et al. [J. Neurochem.
  • Methodology as used by Begley et al. includes: (1) measuring the brain uptake index (BUI) with the equation for a tritiated agent compound:
  • BUI [(brain 3 H/brain 1 C) / (injectate 3 H/ injectate 14 C)] X 100 where the l4 C reference compound is 14 C butanol or an analogous solvent;
  • any of the potential agents and targets for the potential agents ⁇ e.g., calcineurin) or DARPP-32 (such as 3 Pthr 4 phosphorylated DARPP-32) can be labeled.
  • Suitable labels include enzymes, fluorophores ⁇ e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu + , to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands ⁇ e.g., biotin), and chemiluminescent agents.
  • fluorophores ⁇ e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu + , to name a few fluorophores
  • chromophores radioisotopes
  • chelating agents dyes, colloidal gold, latex particles, ligands ⁇ e.g., biot
  • radioactive label such as the isotopes 3 H, l4 C, 32 P, 35 S, 36 C1, 5l Cr, "Co, 58 Co, 59 Fe, 90 Y, 125 1, 1 I I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • Direct labels are one example of labels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. U.V. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6- phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • Other labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • proteins can be labeled by metabolic labeling. Metabolic labeling occurs during in vitro incubation of the cells that express the protein in the presence of culture medium supplemented with a metabolic label, such as [ 35 S]-methionine or [ 32 P]- orthophosphate.
  • a metabolic label such as [ 35 S]-methionine or [ 32 P]- orthophosphate.
  • the invention further contemplates labeling with [ 14 C]-amino acids and [ 3 H]-amino acids (with the tritium substituted at non-labile positions).
  • the component or components of a therapeutic composition of the invention may be introduced parenterally, topically, or transmucosally, e.g., orally, nasally, or rectally, or transdermally.
  • administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome [see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.]. To reduce its systemic side effects, this may be a preferred method for introducing the agent.
  • the therapeutic compound can be delivered in a controlled release system.
  • the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used [see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al, Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med. 321:574 (1989)].
  • polymeric materials can be used [see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida ( 1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al, Science 228:190 (1985); During et al, Ann. Neurol 25:351 (1989); Howard et al, J. Neurosurg. 71:105 (1989)].
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose [see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].
  • Other controlled release systems are discussed in the review by Langer [Science 249:1527- 1533 (1990)].
  • compositions in yet another aspect of the present invention, provided are pharmaceutical compositions of the above. Such pharmaceutical compositions may be for administration for injection, or for oral, topological, nasal or other forms of administration.
  • pharmaceutical compositions comprising effective amounts of the agents of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content ⁇ e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents ⁇ e.g., Tween 80, Polysorbate 80), anti-oxidants ⁇ e.g., ascorbic acid, sodium metabisulfite), preservatives ⁇ e.g., Thimersol, benzyl alcohol) and bulking substances ⁇ e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used.
  • buffer content e.g., Tris-HCl, acetate, phosphate), pH and ionic strength
  • additives such as detergents and solubilizing agents ⁇ e.g., Tween 80, Polysorbate 80), anti-oxidants ⁇ e.g.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Oral Delivery Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers ⁇ e.g., U.S. Patent No. 5,013,556).
  • the formulation will include the agent and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is useful.
  • cellulose acetate trimellitate hydroxypropylmethylcellulose phthalate
  • HPMCP 50 hydroxypropylmethylcellulose phthalate
  • HPMCP 55 polyvinyl acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • Eudragit L30D Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac.
  • CAP cellulose acetate phthalate
  • Shellac Shellac
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
  • the therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression.
  • Colorants and flavoring agents may all be included.
  • the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP polyvinyl pyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives which potentially enhance uptake of the agent are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Controlled release oral formulation may be desirable.
  • the agent could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation.
  • Some enteric coatings also have a delayed release effect.
  • Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.
  • Oros therapeutic system Alza Corp.
  • coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
  • the therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
  • the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
  • the second group consists of the enteric materials that are commonly esters of phthalic acid.
  • Film coating may be carried out in a pan-coater or in a fluidized bed or by compression coating.
  • Nasal Delivery Nasal delivery of the agent is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • the dosage of FK506 for an adult human would be between the range of 0.075 mg/Kg/12 hours to 0.15 mg/Kg/12 hours, where the value indicates the mg quantity of FK506 per Kg weight of the human subject for treatment over a 12 hour period.
  • dosage may be lower.
  • the dosing schedule may vary, depending on the circulation half-life, and the formulation used.
  • Dopamine has been shown to stimulate phosphorylation of DARPP-32, a phosphoprotein highly enriched in medium-sized spiny neurons of the neostriatum. The contribution of
  • Dl-like and D2-like dopamine receptors in the regulation of DARPP-32 phosphorylation has been investigated in mouse striatal slices ⁇ see below). Indeed, one aim of the present study was to determine the relative contribution of Dl-like and D2-like dopamine receptors to dopamine signaling through the DARPP-32/protein phosphatase- 1 cascade, the possible biochemical pathways involved, and the possible effect on this signaling cascade of a widely used antipsychotic drug, raclopride. Dl-like and D2-like receptors were found to have opposing effects on the state of DARPP-32 phosphorylation. The Dl receptor agonist SKF82526 increased DARPP-32 phosphorylation.
  • D2 receptor agonist quinpirole decreased basal as well as the Dl agonist-, forskolin-, and 8-bromo-cAMP-stimulated phosphorylation of DARPP-32.
  • the ability of quinpirole to decrease Dl -stimulated DARPP-32 phosphorylation was found to be calcium-dependent and was blocked by the calcineurin inhibitor, cyclosporin A, suggesting that the D2 effect involved an increase in intracellular calcium and activation of calcineurin.
  • Each slice was placed in a polypropylene incubation tube with 2 ml of fresh Krebs-HC0 3 - buffer containing adenosine deaminase (10 ⁇ g/ml). The slices were preincubated at 30°C under constant oxygenation with 95% 0 2 /5% C0 2 for 60 minutes. The buffer was replaced with fresh Krebs-HC0 3 - buffer after 30 minutes of preincubation.
  • slices were incubated in Ca 2 Xfree/EGTA medium ( 124 mM NaCl, 4 mM KC1, 26 mM NaHC0 3 , 1.25 mM KH 2 PO 4 , 3.0 mM MgS0 4 , 10 mM D-glucose and 1 mM EGTA, pH 7.4) for 20 minutes after 60 minutes of preincubation in Krebs-HC0 3 - buffer.
  • Ca 2 Xfree/EGTA medium 124 mM NaCl, 4 mM KC1, 26 mM NaHC0 3 , 1.25 mM KH 2 PO 4 , 3.0 mM MgS0 4 , 10 mM D-glucose and 1 mM EGTA, pH 7.4
  • Drugs were obtained from following sources: Quinpirole, raclopride, SCH23390, NMDA and MK-801 from Research Biochemicals, Inc.; calyculin A, forskolin, and thapsigargin from LC laboratories; 8-bromo-cAMP from Sigma Chemical Co. After the drug treatment, slices were transferred to Eppendorf tubes, frozen on dry ice, and stored at -80 °C until assayed.
  • the membranes were immunoblotted using a monoclonal antibody (mAb-23; 1:750 dilution) [Snyder et al, (1992)], which is a phosphorylation state-specific antibody raised against a DARPP-32 peptide containing phospho-thr 34 , the site phosphorylated by cAMP-dependent protein kinase.
  • mAb-23 1:750 dilution
  • a monoclonal antibody (C24-5a; 1:7500 dilution) generated against DARPP-32 (Hemmings and Greengard, 1986), which is not phosphorylation state-specific, was used to estimate the total amount of DARPP-32 in samples. None of the experimental manipulations used in the present study altered the total amount of DARPP-32.
  • Antibody binding was revealed by incubation with goat anti-mouse horseradish peroxidase-linked IgG (1:6000-8000 dilution) (Pierce) and the ECL immunoblotting detection system (Amersham). Chemiluminescence was detected by autoradiography using DuPont NEN autoradiography film, and phospho-DARPP-32 bands were quantified by densitometry using a Bio-Rad model 620 video densitometer and Bio-Rad 1-D Analyst software.
  • DARPP-32 determined using the DARPP-32 antibody, C24-5a, was similar in each sample.
  • the phosphorylation state-specific antibody for thr 34 -phosphorylated DARPP-32 also detects the uVXphosphorylated form of inhibitor- 1, a protein phosphatase- 1 inhibitor which is closely related structurally and functionally to DARPP-32 [Aitkin et al, (1982); Williams et al, (1986)]. Although detectable levels of inhibitor- 1 are expressed in medium-sized spiny neurons of the neostriatum, the level of thr ⁇ -phosphorylated inhibitor- 1 was below the sensitivity of detection in both control and dopamine-treated samples.
  • D2-like dopamine receptors might increase the activity of calcineurin, a calcium/calmodulin-dependent protein phosphatase, which has been shown to dephosphorylate phospho-DARPP-32 [Halpain et al, (1990); King et al, (1984)].
  • calcineurin a calcium/calmodulin-dependent protein phosphatase
  • NMD A receptors Effect ofD2 agonist on the level of phosphorylated DARPP-32 is not mediated through the NMD A receptor:
  • the activation of NMD A receptors decreases the phosphorylation of DARPP-32 in the neostriatum (Halpain et al, 1990). This effect has been hypothesized to occur through a mechanism involving increased intracellular calcium and activation of calcineurin.
  • the D2 -mediated regulation of DARPP-32 phosphorylation was examined to determine whether it requires the activation of NMD A receptors.
  • Slices were preincubated in the absence or presence of cyclosporin A (CyA) (5 ⁇ m) for 60 min, followed by the addition of SKF82526 and/or quinpirole.
  • CyA cyclosporin A
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control.
  • TTX like that of MK-801, supports the possibility that, in our preparation, the NMDA receptor is tonically active and dephosphorylates DARPP-32 through activation of calcineurin.
  • the data provide no support for the possibility that quinpirole achieved its effect by release of a neurotransmitter from interneurons.
  • Neostriatal slices were incubated for a total of 20 min in the absence or presence of the NMDA receptor antagonist MK801 (lOO ⁇ m).
  • MK801 was added at 0 min, NMDA (lOO ⁇ m) or quinpirole (l ⁇ m) at 10 min, and SKF82526 (l ⁇ m) at 15 min of incubation.
  • the amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent means ⁇ SEM for four to seven experiments.
  • Neostriatal slices were incubated for a total of 20 min in the absence or presence of TTX (l ⁇ m). TTX was added at 0 min, quinpirole (l ⁇ m) at 10 min, and SKF82526 (l ⁇ m) at 15 min of incubation. The mount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with TTX alone. Data represent means ⁇ SEM for six to seven experiments.
  • Adenylyl cyclase activity in the neostriatum is regulated through the opposing interactions of Dl and D2 receptors (Stoof and Kebabian, 1981). The inhibition of adenylyl cyclase activity by D2 receptors almost certainly contributes to the ability of quinpirole to reduce the phosphorylation of DARPP-32.
  • Calcium omission induced a dramatic increase in the level of phosphorylated DARPP-32 in neostriatal slices and blocked D2-mediated inhibition of DARPP-32 phosphorylation, further supporting a role for the calcium-dependent phosphatase in DARPP-32 regulation.
  • Calcium omission increased DARPP-32 phosphorylation much more than did cyclosporin A treatment. This difference in effectiveness might be due to incomplete inhibition of calcineurin by cyclosporin A.
  • other calcium-dependent processes could also contribute to the regulation of DARPP-32 phosphorylation.
  • a major subtype of adenylyl cyclase is enriched in neostriatum (Cooper et al. , 1995) and calcium omission would be expected to increase the activity of this enzyme, resulting in a potentiation of Dl -stimulated DARPP-32 phosphorylation.
  • the degradation of cAMP in neostriatum is mediated by a calcium-dependent phosphodiesterase activity (Polli and Kincaid, 1994) which would be anticipated to decrease under conditions of low calcium availability, leading to an additional mechanism for the potentiation of Dl -stimulated DARPP-32 phosphorylation.
  • physiological conditions which reduce intracellular calcium would be expected to decrease the driving force for calcium-dependent dephosphorylation and may also affect multiple signaling enzymes within neostriatal neurons to promote cAMP-dependent phosphorylation of DARPP-32 by PKA.
  • D2 receptors on DARPP-32 phosphorylation is calcium-dependent and mediated by an increase in intracellular Ca 2+ and an activation of calcineurin. It is possible that D2 receptors located on DARPP-32-containing neostriatal neurons directly mediate an increase in intracellular Ca 2+ either through regulation of potassium channels, calcium channels or phospholipase C. The contributions of these various signal transduction pathways to the regulation of intracellular Ca 2+ , calcineurin activity and DARPP-32 phosphorylation remain to be clarified.
  • D2-like receptors including the D3 and D4 receptors, which are expressed in medium spiny neurons, albeit at low density (Surmeier et al., 1996), also contribute to signaling pathways responsible for calcineurin-dependent dephosphorylation of DARPP-32.
  • D2 agonists in causing the dephosphorylation of DARPP-32 might be attributable either to a direct effect on Dl receptor-containing neurons or to an indirect effect involving release of neurotransmitter from other neurons.
  • Dl receptor-containing neurons In support of a direct action, Surmeier et al. (1996) have reported that as many as 60% of the neostriatal neurons that contain Dl -class receptors also contain D2-class receptors.
  • the present data indicating that quinpirole induces a 40% decrease in SKF 82526-induced phosphorylation of DARPP-32, is consistent with a limited expression of D2- class receptors in Dl receptor-containing neurons.
  • NMDA receptors which are ubiquitous on medium spiny neurons (Ghasemzadeh et al., 1996) induces a 100% decrease in SKF-induced phosphorylation of DARPP-32 (Table 2).
  • D2 and NMDA receptors may explain the greater efficacy of NMDA in reducing Dl -stimulation of DARPP-32 phosphorylation.
  • NMDA receptors like that of D2 receptors, decreases basal and Dl -stimulated DARPP-32 phosphorylation in neostriatal slices through stimulation of calcineurin, these effects are independent, since the D2 receptor effect was not blocked by MK801, an NMDA receptor antagonist.
  • Dopamine is an important neurotransmitter in the brain. It regulates many basic neuronal functions including motor control, hormonal secretion, motivation and various aspects of cognition. Dopamine is released at presynaptic terminals and functions by binding to post-synaptic dopamine receptors on the cell surface of so called "dopaminoceptive" neurons. Dopaminoceptive neurons are found concentrated in particular regions of the brain most notably the striatum, cortex and hypothalamus. There are five known dopamine receptors which can be grouped into two main classes: Dl-like and D2-like. The binding of dopamine to the various dopamine receptors initiates changes in various signal transduction pathways in the post-synaptic neuron.
  • Example 1 demonstrates that activation of the D2 receptor strongly reduces both the basal level of DARPP-32 phosphorylation and phosphorylation stimulated by Dl agonists ( Figure 3). This decrease in DARPP-32 phosphorylation occurs via the activation of the protein phosphatase calcineurin. Data in Table 1 demonstrate that inhibition of the phosphatase calcineurin using, the drug cyclosporin A, raises the phosphorylation state of DARPP-32. Cyclosporin A also prevents the D2-mediated decrease in DARPP-32 phosphorylation.
  • Example 1 also demonstrates that raclopride, a widely used antipsychotic, increases the phosphorylation of DARPP-32. Such a finding suggests that antipsychotic drugs may act by increasing DARPP-32 phosphorylation. Additional data also supports this premise: (1) Studies examining the level of DARPP-32 in post-mortem samples from 14 schizophrenic patients along with their age, gender and autolysis time-matched controls have shown significant decreases in DARPP-32 in the dorsolateral prefrontal cortex of schizophrenic patients relative to the controls ( Figure 10). These data suggest that the reduction of
  • DARPP-32 phosphorylation and the consequential regulation of protein phosphatase- 1 that DARPP-32 phosphorylation provides is intimately related to the pathophysiology of schizophrenia.
  • Dl antagonists like D2 agonists would be expected to inhibit increases in DARPP-32 phosphorylation.
  • Chambers that are used to assess startle are housed in a sound-attenuated room with a 60 dB(A) ambient noise level. Such chambers consist of a Plexiglass cylinder 8.2 cm in diameter resting on a 12.5 X 25.5 cm Plexiglass frame within a ventilated enclosure. Acoustic noise bursts are presented via a speaker mounted 24 cm above the animal. A piezoelectric accelerometer mounted below the Plexiglass frame detects and transduces motion within the cylinder. The delivery of acoustic stimuli is controlled by a microcomputer. Startle amplitude is defined as the average of 100 readings.
  • the test session consists of giving: (i) a startle stimulus alone (a 118 dB [A] 40 ms broad band burst); or (ii) no stimulation; or (iii) a startle stimulus preceded 100 ms earlier by a prepulse (a 70 dB [A] 20 ms broad band burst).
  • the amount of PPI is expressed as the percentage decrease in the startle response caused by the presentation of the prepulse.
  • the amount of PPI is calculated using the following equation: [(startle amplitude caused by pulse alone minus startle amplitude caused by pulse preceded by prepulse)/(startle amplitude caused by pulse alone)] X 1OO.
  • FK506 is available from Fujisawa Pharmaceuticals.
  • the DARPP-32 knockout mice were prepared as described in U.S. Patent Number 5,777,195 issued July 8, 1998 the Specification of which is hereby inco ⁇ orated by reference in its entirety.
  • Prepulse inhibition (PPI) of the startle reflex refers to the reduction of the startle response due to the prior presentation of a stimulus that is below startle threshold. This effect is presumed to reflect sensorimotor gating mechanisms (Braff and Geyer, 1990). PPI is of particular interest to clinical researchers because it is deficient in schizophrenia [Braff et al., (1992); Grillon et al., (1992)]. Theoretically, a disturbance in such gating mechanisms may permit the intrusion of unwanted sensory information, behaviors or thoughts into ongoing behavioral patterns and may manifest itself as clinical syndromes (Swerdlow et al. , 1993). PPI is disrupted in rodents by treatment with agonists for D2 receptors [Yan et al., (1996); Geyer and Swerdlow, 1994).
  • FK506 is chosen in these studies because it has been shown that cyclosporin fails to cross the blood-brain barrier effectively (Begley et al. , 1990) whereas FK506 effectively crosses the blood-brain barrier.
  • PPI is measured in mice after the following drug treatments: (1) no treatment; (2) after administration of a D2 agonist, such as quinpirole at 0 to 5.0 mg/Kg subcutaneously, preferably between 0.5 to 3.0 mg/Kg, and using 0 mg/Kg as a control if desired; and (3) after administration of D2 agonist and FK506 concurrently (FK506 is administered intravenously in doses ranging from 0.1 mg/Kg to 7.5 mg/Kg, preferably 0.25 mg/Kg to 1 mg/Kg, and using 0 mg/Kg as a control if desired).
  • D2 agonist such as quinpirole at 0 to 5.0 mg/Kg subcutaneously, preferably between 0.5 to 3.0 mg/Kg, and using 0 mg/Kg as a control if desired
  • FK506 is administered intravenously in doses ranging from 0.1 mg/Kg to 7.5 mg/Kg, preferably 0.25 mg/Kg to 1 mg/Kg
  • DARPP-32 a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase- 1. Nature 310: 503-505.

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Abstract

L'invention concerne la régulation bidirectionnelle de la phosphorylation de la DARPP-32 par la dopamine et par les récepteurs D1 et D2 de la dopamine. Cette invention ouvre la voie à de nouveaux traitements de la schizophrénie et des troubles connexes. De plus, ladite invention concerne des procédés permettant d'identifier des agents susceptibles de convenir pour ce type de traitement.
PCT/US1998/022127 1997-10-17 1998-10-19 Traitement de la schizophrenie par utilisation d'un inhibiteur de la dephosphorylation de la darpp-32 WO1999020273A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6989362B1 (en) 1999-10-15 2006-01-24 The Rockefeller University Methods of treating dopamine dysregulation using agents that regulate phosphorylation/dephosphorylation in dopamine signaling
WO2003021225A2 (fr) * 2001-08-31 2003-03-13 The Rockefeller University Procede de classification de medicaments antipsychotiques
WO2003021225A3 (fr) * 2001-08-31 2004-07-08 Univ Rockefeller Procede de classification de medicaments antipsychotiques
EP1575916A2 (fr) * 2001-08-31 2005-09-21 The Rockefeller University Activite de phosphodiesterase et regulation de la signalisation a mediation par phosphodiesterase 1-b dans le cerveau
US7427485B2 (en) 2001-08-31 2008-09-23 The Rockefeller University Method for classification of anti-psychotic drugs
EP1575916A4 (fr) * 2001-08-31 2009-07-29 Univ Rockefeller Activite de phosphodiesterase et regulation de la signalisation a mediation par phosphodiesterase 1-b dans le cerveau
US9157906B2 (en) 2001-08-31 2015-10-13 The Rockefeller University Phosphodiesterase activity and regulation of phosphodiesterase 1B-mediated signaling in brain
EP2270197A3 (fr) * 2002-03-26 2011-02-16 Massachusetts Institute of Technology Cibles, procédés et réactifs pour le diagnostic et le traitement de la schizophrénie
EP1756144A2 (fr) * 2004-05-19 2007-02-28 Merck & Co., Inc. Molecules d'acides nucleiques isolees codant une nouvelle phosphoproteine-darpp-32, proteine codee et utilisations associees
EP1756144A4 (fr) * 2004-05-19 2008-08-20 Merck & Co Inc Molecules d'acides nucleiques isolees codant une nouvelle phosphoproteine-darpp-32, proteine codee et utilisations associees
US9605041B2 (en) 2009-08-05 2017-03-28 Intra-Cellular Therapies, Inc. Regulatory proteins and inhibitors

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