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WO1999020315A1 - Techniques de selection des antipsychotiques atypiques potentiels - Google Patents

Techniques de selection des antipsychotiques atypiques potentiels Download PDF

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WO1999020315A1
WO1999020315A1 PCT/US1998/022492 US9822492W WO9920315A1 WO 1999020315 A1 WO1999020315 A1 WO 1999020315A1 US 9822492 W US9822492 W US 9822492W WO 9920315 A1 WO9920315 A1 WO 9920315A1
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nmda
pcp
induced
apds
response
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Rex Y. Wang
Xiaofu Liang
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Research Foundation of the State University of New York
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention is in the fields of pharmacology and psychiatry. It relates to methods of screening potential atypical antipsychotic drugs (APDs).
  • APDs atypical antipsychotic drugs
  • Schizophrenia represents perhaps the greatest public health concern in psychiatry, affecting approximately 1% of the general population. It is one of the most costly disorders in medicine. It has been estimated that in the USA, the combination of loss of productive employment and direct treatment costs totaled approximately $65 billion dollars in 1991 (Wyatt et al., "An economic evaluation of schizophrenia- 1991" Soc
  • schizophrenia is a disorder of complex cognition and affect. Recent studies have concluded that schizophrenia involves extensive neuropsychological compromise (see Gold and Weinberger, "Cognitive deficits and the neurobiology of schizophrenia” Current Opin Neurobiol 5:225-230. 1995 for review). Clinical and experimental research have provided anatomical, pharmacological, and behavioral evidence for a prominent prefrontal dysfunction in schizophrenia (see Goldman-Rakic and Selemon, "Functional and anatomical aspects of prefrontal pathology in schizophrenia" Schizophrenia Bull 23 :437-458, 1997 for review). Schizophrenia is a mental illness for which present therapy leaves much to be desired.
  • Classical APDs which are presumed to act by blockade of dopamine (DA) D 2 receptors, are useful for the treatment of the positive symptoms, e.g., hallucination, delusion, thought disorder etc.
  • typical APDs often produce various motor side effects, e.g. Parkinsonian symptoms, dystonia, tardive dyskinesia etc.
  • the prototype atypical APD clozapine has been shown to be more efficacious than classical APDs in treating schizophrenics (Kane et al, "Clozapine for the treatment-resistant schizophrenic: A double-blind comparison versus chlorpromazine/benztropine," Arch- Gen. Psychiat.
  • schizophrenic patients are resistant to treatment with neuroleptics (typical APDs), suggesting that other neurotransmitter systems may have a pathogenetic role in these patients.
  • neuroleptics are only partially effective in alleviating the negative, or deficit, symptoms of schizophrenic patients, particularly after resolution of the acute phase of the illness.
  • PET studies of clozapine also failed to support the DA hypothesis of schizophrenia.
  • clozapine's atypical clinical profile may be explained by its relatively high 5-HT 2A /D 2 ratio of pK* value (Meltzer et al, "Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-l, D-2 and serotonin 2 pK- values," J Pharmacol Exp Ther 251 : 238-246, 1989). Indeed, Meltzer et al.
  • NMDA receptor antagonists produce an exacerbation of psychotic symptoms and cognitive impairment (Javitt and Zukin, 1991; Lahti et al., "Ketamine activates psychosis and alters limbic blood flow in schizophrenia” Neuroreport 6: 869-872, 1995; Malhotra et al., "Ketamine-induced exacerbation of psychotic symptoms and cognitive impairment in neuroleptic-free schizophrenics"
  • NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: Modulation by dopamine," J Neurosci 16: 373- 379, 1996; Boyce et al., "Psychomotor activity and cognitive disruption attributable to NMDA, but not sigma, interactions in primates” Behav Brain Res 42:115-121, 1991; Jentsch et al., “Enduring cognitive deficits and cortical dopamine dysfunction in monkeys after long-term administration of phencyclidine” Science 277:953-955, 1997a).
  • APDs are capable of reversing the PCP-induced behavioral changes as determined by in vivo rat behavioral models.
  • the potency of a series of APDs in blocking PCP-induced hyperlocomotion correlated with their affinity for 5-HT 2A receptors (Gleason et al., "Blockade of phencyclidine-induced hyperlocomotion by olanzapine, clozapine and serotonin receptor subtype selective antagonists in mice," Psychopharmacology. 129:79-
  • clozapine and NRA0045 have been shown to improve PCP-induced impairment of performance in a water maze (Okuyama et al., "The atypical antipsychotic profile of NRA0045, a novel dopamine D 4 and 5-hydroxytryptamine 2A receptor antagonist, in rats," Br. J. Pharmacol.. 121:515-525, 1997).
  • atypical APDs might be more effective than typical APDs in reversing non- competitive NMDA receptor antagonist (e.g., PCP, ketamine, MK-801)-induced effects.
  • NMDA receptor hypofunction (NRH) neurotoxicity induced by NMDA antagonist drugs in laboratory animals are also likely to be effective in treating similar types of NRH-related neurotoxicity.
  • This NRH-related neurotoxicity can occur as a component of idiopa hic psychotic illnesses, such as schizophrenia.
  • mPFC medial prefrontal cortex
  • clozapine and amperozide preferentially increase c-fos expression in the mPFC.
  • atypical antipsychotic drugs on striatal Fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Molec Cell Neurosci 3:332-341, 1992; Nomikos et al., "The putative atypical antipsychotic drug amperozide preferentially increases c-fos expression in rat medial prefrontal cortex and lateral septum.”
  • clozapine but not haloperidol or sulpiride, potently antagonizes the action of the 5-HT 2A 2C receptor agonist l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in pyramidal cells of the mPFC (Ashby and Wang, "Effects of antipsychotic drugs on 5-HT 2 receptors in the medial prefrontal cortex: microiontophoretic studies" Brain Research 506:346-348, 1990).
  • DOI 5-HT 2A 2C receptor agonist l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
  • haloperidol is less potent and less efficacious than clozapine in the potentiating action.
  • clozapine, but not haloperidol markedly potentiates glutamate neurotransmission elicited by electrical stimulation of the forceps minor (white matter) in the mPFC.
  • APDs are an object of the present invention to provide methods of screening compounds and predicting their therapeutic efficacy as APDs in treating not only positive but also deficit symptoms in schizophrenia.
  • the present invention is an in vitro method of screening and assessing compounds for potential efficacy as atypical APDs.
  • One method of the present invention comprises measuring the ability of a compound (i.e., potential APD) to prevent or inhibit the blockade of the NMDA receptor-ionophore complex in pyramidal neurons caused by a non-competitive NMDA receptor antagonist.
  • a compound i.e., potential APD
  • An example of a non-competitive NMDA receptor antagonist to be utilized is PCP.
  • the neurons are, preferably, pyramidal neurons of the mPFC of a mammal.
  • the ability of a compound to prevent the blockade of the NMDA receptor by the non-competitive NMDA antagonist is determined with electrophysiological techniques.
  • the method of the present invention includes screening a compound for potential efficacy as an atypical APD, which comprises treating pyramidal neurons with the compound; treating the pyramidal neurons with an effective amount of a non-competitive NMDA antagonist capable of blocking the NMDA receptor channels in untreated pyramidal neurons; subsequently administering to the neurons a response- inducing amount of NMDA; and measuring the NMDA-induced response of the pyramidal neurons.
  • an in vitro method of screening and assessing the atypical antipsychotic potential of a compound is accomplished by measuring the ability of the compound to reverse the blockade of the NMDA receptor-ionophore complex in pyramidal neurons caused by a non-competitive NMDA receptor antagonist.
  • An example of a non-competitive NMDA receptor antagonist to be utilized is PCP.
  • the neurons are, preferably, pyramidal neurons of the mPFC of a mammal.
  • the ability of a compound to reverse the induced blockade of the NMDA receptor by the non-competitive NMDA antagonist is determined with electrophysiological techniques.
  • the method of screening a compound for potential efficacy as an atypical APD includes treating pyramidal neurons with an effective amount of a non- competitive NMDA antagonist capable of blocking the NMDA receptor channels in untreated pyramidal neurons; treating the pyramidal neurons with the compound; subsequently administering to the neurons a response-inducing amount of NMDA; and measuring the NMDA-induced response of the pyramidal neurons.
  • the method of screening a compound for potential efficacy as an atypical APD includes administering a potential atypical APD to a mammal; thereafter administering the potential atypical APD plus an effective amount of non-competitive NMDA antagonist to the mammal; subsequently administering a response-inducing amount of NMDA and AMP A to pyramidal neurons of the mammal; and measuring the NMDA and AMPA-induced response of the pyramidal neurons.
  • the NMDA and AMPA-induced response of the pyramidal neurons is measured electrophysiologically by an inward current.
  • the method of screening a compound for potential efficacy as an atypical APD includes measuring the ability of a compound (i.e., potential APD) to prevent or inhibit the effects produced by subchronic treatment with a non-competitive NMDA receptor antagonist such as PCP.
  • a compound i.e., potential APD
  • PCP non-competitive NMDA receptor antagonist
  • the neurons are, preferably, pyramidal neurons of the mPFC of a mammal.
  • the ability of a compound to prevent the blockade of the NMDA receptor by the non-competitive NMDA antagonist is determined with electrophysiological techniques.
  • the present invention provides reliable in vitro methods of screening compounds with a profile of atypical APDs. Unlike animal behavioral models, more precise criteria are utilized to screen potential compounds with the present invention. In addition, the methods of the present invention avoids the problems associated with live animal testing.
  • Panels A-C are current traces of NMDA induced responses in mPFC pyramidal neurons: Panel A - shows that the administration of PCP 5 ⁇ M into the artificial cerebrospinal fluid (ACSF) for 5 minutes in partially blocked the first NMDA response and completely blocked the second NMDA response, reflecting the use- dependent blockade of the NMDA receptor-ionophore by PCP; Panel B - shows that pretreatment with 100 nM Ml 00907 for 15 minutes prior to PCP administration completely prevented the PCP induced blockade of the NMDA response; and Panel C - shows that the administration of Ml 00907 following PCP administration strikingly shortened the duration of the blockade of NMDA receptor channel caused by PCP.
  • a - shows that the administration of PCP 5 ⁇ M into the artificial cerebrospinal fluid (ACSF) for 5 minutes in partially blocked the first NMDA response and completely blocked the second NMDA response, reflecting the use- dependent blockade of the NMDA receptor-iono
  • Panels A-C are plot graphs of the NMDA induced response as a function of time in mPFC pyramidal neurons: Panel A - shows that PCP reduces and blocks NMDA-induced inward current in a concentration-dependent manner; Panel B - shows that Ml 00907, in a concentration-dependent fashion, either prevents totally or reduces markedly the ability of PCP to block NMDA-induced inward current; and Panel
  • FIG. 3 Panels A-E, are current traces of NMDA induced responses in mPFC pyramidal neurons to illustrate that pretreated with either Ml 00907 (100 nM), clozapine
  • Figure 4 is a plot graph comparing NMDA induced responses as a function of time in mPFC pyramidal neurons pretreated with 100 nM concentrations of Ml 00907, clozapine, olanzapine, haloperidol, raclopride, Ml 00009, or 10 nM olanzapine, followed by administration 1 ⁇ M PCP.
  • Panel A illustrates current traces of 5 and 10 ⁇ M AMP A and 10 and 20 ⁇ M NMDA in slices from vehicle- and PCP-treated rats.
  • Panel B is a graphic comparison of NMDA-o / AMPA 5 and NMDA 20 / AMPA 5 response ratios (measured as the peak amplitude of currents induced by 10 and 20 ⁇ M NMDA over current induced by 5 ⁇ M AMP A) in slices from control (circles) and PCP-treated (triangles) rats.
  • Panel C illustrates traces of averaged EPSCs evoked by 60 consecutive paired-stimuli in slices from control (in row 1) and PCP-treated (in row 2) rats.
  • atypical APDs are capable of preventing or reversing PCP-induced effects on pyramidal cells of the mPFC. Accordingly, this invention provides methods for screening potential atypical APDs, i.e., measuring the ability of compounds to prevent or reverse effects induced by either acute or subchronic treatment with PCP.
  • PCP-induced effects are measured by using electrophysiological techniques including NMDA responses, PPF and EPSC variation, and NMDA receptor- mediated neurotransmission in pyramidal cells of the mPFC in in vitro brain slice preparations.
  • electrophysiological techniques including NMDA responses, PPF and EPSC variation, and NMDA receptor- mediated neurotransmission in pyramidal cells of the mPFC in in vitro brain slice preparations.
  • the ability or efficacy of the compound to either inhibit or reverse PCP- induced effects indicates the potential of the compound as an atypical APD.
  • the more effective the compound is at either inhibiting or reversing the effect of the non- competitive NMDA antagonist on the pyramidal neurons the greater its potential as an atypical APD.
  • a method of screening potential APDs is provided by demonstrating the ability of potential APDs to prevent PCP-induced blockade of NMDA responses in pyramidal neurons of the mPFC.
  • the method of screening potential APDs includes: applying a response-inducing amount of NMDA with a 15 minute inter-application interval to a pyramidal cell recorded intracellularly throughout the experiment (Wang and Liang, 1998). This establishes stable baseline responses of NMDA, then a potential APD is administered in the perfusion solution (artificial cerebrospinal fluid or ACSF), and 30 minutes after the potential APD is administered, an effective amount of non-competitive NMDA antagonist (e.g. 1 ⁇ M PCP) will then be added to the ACSF.
  • a control drug e.g., vehicle control, a typical APD or a non-APD drug
  • the potential APD is applied.
  • a control drug includes vehicle control, a typical APD or a non- APD drug.
  • typical APDs are chlorpromazine, haloperidol, trifluoperazine and other typical APDs.
  • Non-APDs include, but are not limited to, fluoxetine, Ml 00009, morphine, meperidine and other non-antipsychotic drugs.
  • the amount of the potential APD to be administered is variable. However, to obtain relative comparative data, one of ordinary skill in the art should administer a known atypical APD, such as clozapine, to inhibit the effects of the non-competitive NMDA antagonist, from which an equivalent amount of the potential compound can be determined and utilized for assessing efficacy of the compound.
  • a known atypical APD such as clozapine
  • a variable amount of the potential APD administered also includes an amount that establishes the effectiveness of the potential APD to prevent the non- competitive NMDA receptor antagonist effect of PCP. In which case, a concentration- response curve for the potential APD to prevent PCP's blockade of the NMDA response in pyramidal cells of the mPFC is obtained.
  • an effective amount of a non- competitive NMDA antagonist means an amount effective to inhibit NMDA-induced response in unprotected pyramidal neurons (see Wang and Liang, 1998). The actual amount required will vary with the non-competitive NMDA antagonist selected.
  • a preferred antagonist is PCP which can be administered in concentrations of at least 0.5 ⁇ M to about 5 ⁇ M.
  • Other antagonists to be utilized with the invention include, but are not limited to, ketamine, and MK-801.
  • the "response-inducing amount of NMDA” is any amount of NMDA that will elicit a measurable response (e.g., inward current in a voltage-clamp mode or membrane depolarization in a current-clamp mode) in untreated pyramidal neurons.
  • a measurable response e.g., inward current in a voltage-clamp mode or membrane depolarization in a current-clamp mode
  • the actual amount required to elicit a measurable response in pyramidal neurons is variable.
  • One such factor is the location of the neuron and the density of NMDA receptors contained in the soma-dendrites of the neuron and pre-synaptic axon terminals.
  • the response of the pyramidal neurons to NMDA can be measured by any suitable means.
  • the response of the pyramidal neurons to NMDA is measured electro-physiologically. This can be accomplished, for instance, by measuring the NMDA-induced inward current exhibited by the pyramidal neurons through intracellular recording and single-electrode voltage clamp techniques.
  • a method of screening potential APDs is provided by ascertaining the efficacy of the compound to reverse the blockade of the NMDA receptor channels in pyramidal neurons caused by a non-competitive NMDA antagonist. It has previously been demonstrated that the purported atypical APD
  • Ml 00907 administered after PCP will shorten the duration of the blockade of NMDA responses in pyramidal cells of the mPFC (Wang and Liang, 1998).
  • the method for screening the potential atypical APDs includes: establishing the baseline response of NMDA in a pyramidal neuron of the mPFC; administering an effective amount of a non-competitive NMDA antagonist (e.g. 5 ⁇ M PCP) in the ACSF; 30 minutes after the administration of PCP, the potential APD or a control drug (e.g., the vehicle control, a typical APD or a non-APD drug) is administered to the ACSF.
  • a control drug e.g., the vehicle control, a typical APD or a non-APD drug
  • various doses of the potential atypical APD are administered for one week, then the potential atypical APD plus PCP (2 mg/kg, b.i.d.) for the following week, and experiments are then performed 48-60 hours after the last PCP injection (a 48-60 hours withdrawal period).
  • PCP The dose of PCP is selected based upon those of ordinary skill in the art (Jentsch et al., 1997a, b; Sturgeon et al., "Behavioral effects of chronic phencyclidine administration in rats” Psvchopharmacology 76: 52-56, 1982; Verebey et al., "Phencyclidine-induced stereotypy in rats: Effects of methadone, apomorphine, and naloxone.” Psvchopharmacology 75: 44- 47, 1981). Again control drugs are used such that the results can be compared to those of the potential atypical APDs.
  • the method of the present invention includes administering a potential atypical APD to a mammal; thereafter administering the potential atypical APD plus an effective amount of a non-competitive NMDA antagonist to the mammal; subsequently administering a response-inducing amount of NMDA and AMP A to the pyramidal neurons of the mammal; measuring the NMDA and AMPA-induced response of the pyramidal neurons and calculating a ratio of the NMDA and AMPA-induced peak currents.
  • NMDA and AMP A are any amount of NMDA and AMPA that will elicit a measurable response (e.g., inward current in a voltage-clamp mode or membrane depolarization in a current-clamp mode) in pyramidal neurons.
  • a measurable response e.g., inward current in a voltage-clamp mode or membrane depolarization in a current-clamp mode
  • the actual amount required to elicit a measurable response in pyramidal neurons is variable.
  • One such factor is the location of the neuron and the density of NMDA receptors contained in the soma- dendrites of the neuron and pre-synaptic axon terminals.
  • EXAMPLE 1 PREVENTION AND REVERSAL OF ACUTE PCP-INDUCED BLOCKADE OF NMDA RESPONSES IN PYRAMIDAL CELLS OF THE mPFC
  • ACSF cerebrospinal fluid
  • coronal (transverse) slices of mPFC (450 ⁇ M thick) were cut in ice-cold ACSF containing (in mM): NaCl 117, KC1 4.7, CaCl 2 2.5, MgCl 2 1.2, NaHCO 3 25, NaH 2 PO 4 1.2 and D- glucose 11, aerated with 95% O 2 /5% CO 2 (pH 7.4) and kept submerged at room temperature for at least 1 hour to allow for recovery.
  • a single slice was then transferred to a recording chamber (32°C). The chamber was continuously perfused with ACSF at a constant rate of 2 ml/min.
  • NMDA was applied by placing a microdrop (10 ⁇ l) of concentrated solution (1 mM, with a dilution factor 1 : 100) on a marked spot in the inflow channel of the chamber
  • NMDA-evoked response in pyramidal neurons of the rat medial prefrontal cortex slices consists of NMDA and non-NMDA components
  • Brain Research. 768:361-364, 1997; Arvanov and Wang, 1998; Arvanov et al., 1997; Wang and Liang, 1998 Repeated microdrop application of NMDA to the same pyramidal cell with an inter-application interval of approximately 15 minutes produced a consistent inward current, although the baseline current caused by NMDA varied from cell to cell (30 - 200 pA).
  • two or three stable consecutive control responses to NMDA were obtained, the average of which was counted as the baseline of NMDA, prior to any drug tests.
  • PCP-INDUCED BLOCKADE OF NMDA RESPONSES The techniques of intracellular recording and single-electrode voltage-clamp were used to avoid interference of voltage-related alteration of ion channels as bath application of PCP invariably produced a significant membrane hyperpolarization.
  • PCP at concentra- tions of 0.2, 1 and 5 ⁇ M was administered to the ACSF for 5 minutes.
  • NMDA was then administered at an inter-application interval of 15 minutes.
  • the current traces illustrating the blockade caused by the 5 ⁇ M concentration of PCP are shown in Figure 1, Panel A. The first NMDA response was partially blocked and the second NMDA response was completely blocked.
  • Panel B As can be seen from Figure 2, Panel B, concentrations of 20 and 100 nM Ml 00907 significantly reduced and completely abolished PCP's effect, respectively, whereas 4nM Ml 00907 was without effect.
  • Ml 00907 to reverse PCP-induced blockade of the NMDA receptor channel was investigated.
  • Ml 00907 at a concentration of 100 nM was administered following 5 ⁇ M PCP administration.
  • NMDA was administered at an inter-application interval of 15 minutes.
  • Current traces illustrating the reversal of PCP-induced blockade by Ml 00907 are shown in Figure 1 , Panel C.
  • Ml 00009 As a negative control, the ability of Ml 00009 to reverse PCP-induced blockade of the NMDA receptor channel was ascertained. Ml 00009 at a concentration of 100 nM was administered following 5 ⁇ M PCP. NMDA was administered at an inter-application interval of 15 minutes. A comparison of the NMDA-induced response of Ml 00907 and
  • Ml 00009 is graphically depicted in Figure 2, Panel C.
  • Figure 2 Panel C, shows that Ml 00009 does not reverse PCP-induced blockade of the NMDA response, while Ml 00907 significantly reverses PCP-induced blockade of the NMDA response.
  • RESPONSES Following the above described procedure, the ability of 100 nM concentrations of Ml 00907, Ml 00009, clozapine, haloperidol and raclopride to prevent 1 ⁇ M PCP-induced blockade of NMDA responses was investigated. In addition, the ability of 10 nM olanzapine to prevent 1 ⁇ M PCP-induced blockade of NMDA responses was examined. Clozapine was selected since it is a known atypical APD. Olanzapine was selected because it is a purported new atypical APD. Haloperidol was selected since it is a known classical APD. Raclopride was selected because it is a known DA D 23 receptor antagonist.
  • the concentration of 10 nM was selected for olanzapine because it has been demonstrated at this concentration Olanzapine 's potentiation effect on NMDA- induced inward current reached maximum level in pyramidal cells of the mPFC (Liang and Wang, unpublished observations).
  • Current traces illustrating the prophylactic effect, if any, of these compounds are shown in Figure 3, Panels A-E.
  • PCP (2 mg/kg, b.i.d.) for the following week, and sacrificed 48-60 hours after the last PCP injection.
  • the dose of PCP was selected based upon those reported in the literature (Jentsch et al., 1997a, b; Sturgeon et al., 1982; Verebey et al., 1981) and the observation of marked increase in locomotor activity and stereotypy by PCP.
  • clozapine and haloperidol were selected because it was previously demonstrated that subchronic treatment of rats with either clozapine or haloperidol at these doses produced a "depolarization block" of spontaneously active dopamine neurons in the midbrain (White and Wang, "Differential effects of classical and atypical antipsychotic drugs on A9 and AlO dopamine neurons" Science 221 :1054-1057,1983). This is another screening method for APDs.
  • the brain slice offers a number of advantages in conducting the proposed studies.
  • VOLTAGE -CLAMP Standard intracellular and single electrode voltage-clamp recording techniques were used to record pyramidal cells in layers V and VI of the mPFC in slice preparations as described previously by Arvanov and Wang, 1997, 1998; Arvanov et al., 1997; Wang and Liang, 1998. Intracellular recordings were performed using 4 M K-acetate or 3 M KCl-filled microelectrodes (tip resistances 60 - 90 M ⁇ ). The electrophysiological criteria we used for distinguishing presumed pyramidal versus non-pyramidal cells have been previously published (see Arvanov et al., 1997; Arvanov and Wang, 1998; Yang et al.,
  • the pyramidal cells exhibit a longer spike duration ( ⁇ 1 ms at half- maximum spike amplitude) than that of interneurons and show pronounced spike- frequency adaptation in response to constant-current depolarizing pulses.
  • interneurons exhibit a brief duration of their action potentials and lack pronounced spike frequency adaptation.
  • Agonists were applied every 15 minutes by placing a microdrop ( 10 ⁇ l) of concentrated solution (with 1 :100 dilution factor) on a marked spot in the inflow channel (Arvanov et al., 1997; Arvanov and Wang, 1998; Wang and Liang, 1998).
  • the PARADIGM OF PAIRED-PULSE FACILITATION fPPF is considered to be a presynaptic phenomenon, resulting from a transient increase of presynaptic Ca 2+ , caused by a conditioning synaptic response (Hess et al., 1987; Zucker, 1989).
  • variance analysis of the statistical parameter m cv mean 2 /variance (M 2 /F 2 ), the alterations of which have been reported to be closely related to presynaptic sites, was also used.
  • NMDA concentrations of NMDA (10 and 20 ⁇ M) and AMPA (5 and 10 ⁇ M) were chosen based on previous experiments (Arvanov et al., 1997; Arvanov and Wang, 1998; Wang and Liang, 1998) and because they produced a submaximal response. Although it is difficult to generate a complete concentration-response curve for NMDA, because of the difficulty for voltage- clamping the large currents induced by concentrations of NMDA higher then 20 ⁇ M, this normalization of data would minimize daily variations and differential sensitivities among cells. Because PCP should not interact directly with the AMPA subtype of glutamate receptors, AMPA-induced responses were used as a reference. In fact, the AMPA ⁇ o/AMPA 5 ratio was unchanged in all PCP-treated groups, i.e. the ratios were not significantly different from that of the vehicle control group (Table 1).
  • the difference of the PPF values between the two groups was statistically significant (t-test, p ⁇ 0.05).
  • Table 1 Comparison of response ratios for NMDA and AMPA and response to PCP challenge in pyramidal cells of the medial prefrontal cortex from vehicle control and PCP-treated rats.
  • Values are mean ⁇ S.E.M.; n represents number of neurons.
  • the response ratio was obtained using peak amplitude of current induced by NMDA 10 and 20 ⁇ M and AMPA 5 and 10 ⁇ M in each neuron voltage clamped at -60 mV.
  • PCP lwk 0.5h
  • PCP lwk/lwk represents groups in which experiments were performed 0.5 hr, 48-60 hr and 1 week after 1 week subchronic treatment with PCP, respectively.
  • % blockade of NMDA responses produced by PCP challenge we compared NMDA-induced current before and after inclusion of 1 ⁇ M PCP to perfusing ACSF.
  • Cells were held at -65 mV; sAHP was obtained by passing 0.1 nA through the recording microelectrode. b Number of action potentials which were evoked by a depolarizing pulse 0.5 nA, 600 ms; cells were held at
  • HALOPERIDOL ON SUBCHRONIC PCP-INDUCED ALTERATION OF RATIOS OF NMDA/AMPA To determine if antipsychotic drugs may prevent the effects produced by the subchronic PCP treatment, we pretreated rats for one week with either clozapine (25 mg/kg/day, i.p.) or haloperidol (0.5 mg/kg/day, i.p.) alone and the following week co- treatment of either antipsychotic drug with PCP. As shown in Table 3, clozapine prevented the subchronic PCP-induced enhancement of both NMDA 20 /NMDA, 0 and NMDA 20 /AMPA 5 ratios.
  • Table 3 Comparison of response ratios for NMDA and AMPA in pyramidal cells of the medial prefrontal cortex from PCP-, clozapine plus PCP-, and haloperidol plus PCP-treated rats. Responses were obtained using NMDA 10 and 20 ⁇ M and AMPA 5 and 10 ⁇ M.

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Abstract

La présente invention concerne des techniques de sélection des médicaments antipsychotiques (APD) atypiques potentiels. Une de ces techniques consiste à évaluer la capacité des APD potentiels à empêcher le blocage des réponses NMDA dans des cellules pyramidales du cortex préfontal interne dans des préparations en coupe de tissus cérébraux in vitro, ce blocage étant induit par la phencyclidine (PCP), antagoniste des récepteurs N-méthyl-D-aspartate (NMDA). Une autre technique consiste à évaluer la capacité des APD atypiques potentiels à empêcher les effets produits par un traitement répété avec la PCP des cellules pyramidales du cortex préfrontal interne dans des préparations en coupe de tissus cérébraux in vitro. Chez les humains, la PCP ne provoque pas seulement des hallucinations et des délires, mais elle génère également un état apathique associé et une sorte de troubles de la pensée caractéristiques de la schizophrénie. De plus, dans la schizophrénie, les antagonistes des NMDA produisent une exacerbation des symptômes psychotiques et une déficience cognitive. On a par ailleurs observé des déficits cognitifs chez les singes et les rats traités à la PCP. On a largement pu faire la preuve que les APD atypiques sont beaucoup plus efficaces que les APD classiques dans la prévention ou la réversion des effets induits par la PCP. La présente invention concerne de nouvelles techniques électrophysiologiques permettant de sélectionner les APD atypiques potentiels et de prévoir leur efficacité thérapeutique dans les symptômes schizophréniques négatifs et les déficits cognitifs et neuropsychologiques.
PCT/US1998/022492 1997-10-23 1998-10-23 Techniques de selection des antipsychotiques atypiques potentiels Ceased WO1999020315A1 (fr)

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US7132433B2 (en) * 2000-05-25 2006-11-07 Aventis Pharmaceuticals Inc. Use of (+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4- piperidinemethanol or its prodrug in the treatment of behavioral or psychological symptoms associated with a disease
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WO2000079273A3 (fr) * 1999-06-21 2001-05-25 Matsushita Electric Industrial Co Ltd Procedes et dispositif servant a detecter in vitro et a caracteriser, dans un echantillon, des psychotropes, a l'aide d'une analyse de l'activite electrique repetitive
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WO2004084952A1 (fr) * 2003-03-28 2004-10-07 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methode de detection in vivo de ligands des recepteurs dopaminergiques d3.
EP3702448A1 (fr) 2019-03-01 2020-09-02 Neuroproof GmbH Réseau neuronal et procédé de surveillance de l'équilibre excitateur et inhibiteur
WO2020178226A1 (fr) 2019-03-01 2020-09-10 NeuroProof GmbH Réseau neuronal et procédé de surveillance de l'équilibre excitateur et inhibiteur

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