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WO2001087285A2 - Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda - Google Patents

Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda Download PDF

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WO2001087285A2
WO2001087285A2 PCT/US2001/015372 US0115372W WO0187285A2 WO 2001087285 A2 WO2001087285 A2 WO 2001087285A2 US 0115372 W US0115372 W US 0115372W WO 0187285 A2 WO0187285 A2 WO 0187285A2
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psd95
psd93
psd
protein
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WO2001087285A3 (fr
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Roger A. Johns
Yuanxiang Tao
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • MAC minimum alveolar concentration
  • PSD postsynaptic density
  • NMDA N-methyl-D-aspartate
  • One embodiment of the invention provides a method for relieving acute or chronic pain. According to the method an effective amount of an agent which inhibits expression of PSD93 or PSD95 is administered to a subject in need of pain relief. The agent relieves acute or chronic pain experienced by the subject.
  • Another embodiment of the invention provides a method for treating or preventing hyperalgesia.
  • an effective amount of an agent which inhibits expression of PSD93 or PSD95 is administered to a subject who has or is at risk of developing hyperalgesia.
  • the administration relieves or prevents hyperlagesia experienced by the subject.
  • Another aspect of the invention is a method of reducing a threshold for anesthesia.
  • An anesthetic and an agent which inhibits expression of PSD93 or PSD95 are administered to a subject.
  • the amount of anesthetic administered achieves a desired anesthetic effect even though the amount administered is less than the amount required in the absence of the agent to achieve the desired anesthetic effect. This minimizes the serious side effects of the anesthetics including cardo vascular and respiratory depression.
  • the present invention also provides an isolated and purified antisense polynucleotide which is complementary to PSD95 or PSD93 mRNA.
  • Another embodiment of the invention is a method for relieving acute or chronic pain.
  • An effective amount of an agent which inhibits interaction of a first protein selected from the group consisting of PSD93 and PSD95, with a second protein selected from the group consisting of nNOS and NMDA receptor, is administered to a subject in need thereof.
  • the agent does not cause cardiovascular or respiratory depression.
  • the administration relieves acute or chronic pain experienced by the subject.
  • An effective amount of an agent which inhibits interaction of a first protein selected from the group consisting of PSD93 and PSD95, with a second protein selected from the group consisting of nNOS and NMDA receptor, is administered to a patient experiencing hyperalgesia or who is at risk of developing hyperalgesia.
  • the agent does not cause cardiovascular or respiratory depression. Hyperalgesia experienced by the subject is relieved or prevented by the administration.
  • Also provided by the present invention is a method of reducing a threshold for anesthesia.
  • An anesthetic and an agent which inhibits interaction of a first protein selected from the group consisting of PSD93 and PSD95, with a second protein selected from the group consisting of nNOS and NMDA receptor, are administered to a subject.
  • the agent does not cause cardiovascular or respiratory depression.
  • the amount of anesthetic administered is less than the amount required in the absence of the agent to achieve a desired anesthetic effect. The desired anesthetic effect is thus achieved.
  • the present invention also provides a method of anesthetizing a subject.
  • An agent which inhibits expression of PSD93 or PSD95 is administered to a subject.
  • the agent renders the subject unconscious or sedated.
  • Another embodiment of the invention provides a method of anesthetizing or sedating a subject.
  • An agent which inhibits interaction of a first protein selected from the group consisting of PSD93 and PSD95, with a second protein selected from the group consisting of nNOS and NMDA receptor, is administered to a patient.
  • the agent does not cause cardiovascular or respiratory depression.
  • the agent renders the subject unconscious or sedated.
  • Yet another aspect of the invention is a method of screening for substances useful for relieving pain or inducing unconsciousness or sedation.
  • a test substance is contacted with a first protein and a second protein under conditions where the first protein and the second protein bind to each other.
  • the first protein is selected from the group consisting of PSD93, PSD95, and a combination thereof.
  • the second protein is selected from the group consisting of nNOS, NMDA receptor, NR2A subunit, NR2B subunit, and combinations thereof.
  • the mixture of proteins is assayed to determine the binding of the first protein to the second protein. Any parameter which reflects that binding can be assayed.
  • Such parameters include the amount of free nNOS, the amount of free PSD93, the amount of free PSD95, the amount of free NMDA receptor, the amount of free NR2A subunit, the amount of free NR2B subunit, the amount of bound nNOS, the amount of bound PSD93, the amount of bound PSD95, the amount of bound NMDA receptor, the amount of bound NR2A subunit, the amount of bound NR2B subunit and combinations of them.
  • a test substance which increases the amount of free nNOS, free PSD93, free PSD95, free NMDA receptor, free NR2A subunit, or free NR2B subunit, or which decreases the amount of bound nNOS, bound PSD93, bound PSD95, bound NMDA receptor, bound NR2A subunit, or bound NR2B subunit is identified as a candidate drug for relieving pain or inducing unconsciousness or sedation.
  • Fig. IA and IB Expression of PSD-95/SAP90 mRNA and protein in the spinal cord.
  • Fig. 1 Aa immunoblot showing the expression of PSD-95/SAP90 in the PSD fractions of the spinal cord (SC), dorsal root ganglion (DRG) and other brain regions (as positive controls) in the normal rats.
  • HI hippocampus
  • CO cortex
  • CE cerebellum.
  • Fig. lAb immunoblot showing representative effects of PSD-95/SAP90 antisense (AS), missense (MS) and sense (SE) ONDs, as well as saline (SA), on the expression of PSD- 95/SAP90, nNOS and NR2A/2B in the spinal cord.
  • SA saline
  • PC positive control tissue from hippocampus.
  • Asterisk non-specific band by the secondary antibody, useful to control for protein loading and blot exposure times.
  • RT-PCR analysis showed that 0.737 Kb mRNA was detected in the spinal cord and other brain regions (hippocampus, cortex, cerebellum and brainstem), but not in muscle.
  • PCR product was directly cloned into the TA cloning vector and verified as PSD-95/SAP90 by automatic DNA sequencing, ⁇ -actin mRNA was used as a loading control.
  • Fig. 2A and 2B Distribution of PSD-95/SAP90 immunoreactivity in lumbar enlargement segments of the spinal cord.
  • the PSD-95/SAP90 immunoreactivity was localized mainly in lamina I and outer lamina II (A). Under high magnification, many PSD-95/SAP90 immunoreactive puncta were observed (B). Scale bars: 200 ⁇ m in A; 40 ⁇ m in B.
  • Fig. 3 Identification of a ternary complex assembled by PSD-95/SAP90 with NR2A/2B and nNOS in the spinal cord neurons.
  • PSD-95/SAP90 antibody immunoprecipitated not only PSD-95/SAP90 but also NR2A/2B and nNOS.
  • endothelial NOS eNOS
  • Ten ⁇ g protein was loaded in INPUT lane and 100 ⁇ g in other lanes.
  • PSD95 and PSD93 mediate the interaction of NMDA receptors and nNOS in the spinal cord, and are involved in generating responses to painful stimuli.
  • the inventors have found that inhibition of the interaction of NMDA receptors and nNOS via PSD95 and PSD93 can attenuate responses to painful stimuli, as well as lower thresholds for anesthetics.
  • inhalational anesthetics themselves inhibit the interaction of NMDA receptors and nNOS via PSD95 and PSD93.
  • new and improved anesthetics and sedatives can be identified using the identified interaction as an assay system.
  • Acute or chronic pain can be relieved or prevented according to the present invention by administering to a subject an effective amount of an agent which inhibits expression of PSD93 or PSD95.
  • the agents of the present invention also can be used to treat or prevent hyperalgesia, as well as to reduce a threshold for anesthesia.
  • the agent used can be an antisense oligonucleotide (ODN) which is complementary to mRNA encoding PSD93 or PSD95.
  • ODN antisense oligonucleotide
  • the antisense oligonucleotide is complementary to nucleotides encoding a PDZ domain. More preferably the antisense oligonucleotide is complementary to nucleotides 241 to 258 of PSD95.
  • Oligonucleotides useful in the invention can be naked oligonucleotides or can be administered in a vector, liposome, particle or other protective formulation. If in a vector, the vector can express RNA molecules which are complementary to the native PSD93 or PSD95 mRNAs. Also encompassed by the present invention are oligonucleotides which contain nucleotide analogue moieties to render the oligonucleotides less susceptible to enzymatic degradation. Suitable nucleotide analogue moieties are known in the art and include phophorothioates.
  • Agents according to the present invention can be administered any way known in the art which is convenient and efficient for the particular agent and the application.
  • the agent is administered intrathecally, per os, or intravenously.
  • other means can be used as appropriate, including subdermal, subcutaneous, rectal, intraperitoneal, subarachnoid, caudal, epidural, inhalational, and intramuscular administrations.
  • Anesthetics and sedatives used in the methods of the present invention can also be administered by any of these same means.
  • Preferred anesthetics according to the invention are inhalational anesthetics, including halothane, isoflurane, desflurane, xenon, and sevoflurane.
  • compositions are provided for inhibiting expression of PSD95 or PSD93.
  • Such compositions comprises an isolated and purified antisense polynucleotide which is complementary to PSD95 or PSD93 mRNA.
  • the polynucleotide is complementary to nucleotides encoding a PDZ domain. Any of the three such domains can be targeted, although the third such domain, i.e., the C-terminal PDZ domain, may be the most effective.
  • One particular oligonucleotide which has been found to be effective is complementary to nucleotides 241 to 258 of PSD95.
  • the analogous nucleotides of PSD93 can also be used.
  • the polynucleotide can be formulated in a pharmaceutically acceptable vehicle so that it can be used to prevent pain or to lower an anesthetic or sedative threshold.
  • a pharmaceutically acceptable vehicle so that it can be used to prevent pain or to lower an anesthetic or sedative threshold.
  • Particular vehicles which are suitable for intrathecal or inhalational therapy can be advantageously used.
  • the formulations can be in liquid or vapor form. They can be vaporized by bubbling a gas through them.
  • the formulations of the invention will be manufactured under regulatory-approved conditions for administration to humans. Requirements for such formulations typically include sterility and freedom from pyrogens.
  • agents which specifically inhibit the expression of PSD93 or PSD95 be used in the methods of the present invention, but also agents which inhibit the interaction of PDS93 or PSD95 with either nNOS or NMDA receptors.
  • Such agents can be used for the same purposes as discussed above, for relieving acute or chronic pain, for reducing the threshold for anesthetics and sedatives, and for anesthetizing and sedating patients directly.
  • Agents useful according to the present invention do not cause cardiovascular or respiratory depression.
  • Such agents can be administered to the same populations of patients as discussed above, i.e., those in need of anesthesia, those in need of relief from chronic or acute pain, and those who experience hyperalgesia or are at risk of developing hyperalgesia.
  • Such patients include those whose pain is mechanical, thermal, neuropathic, or inflammatory in origin.
  • Protein interaction-inhibitory agents of the invention preferably bind to a PDZ domain of any of the binding participants, including nNOS, NMDA receptors, PDS93 or PDS95.
  • the agent does not impair motor function, i.e., locomotion.
  • Such agents can be identified by any of a number of screening techniques which rely on the inhibition of expression or interactions of PDS93 or PDS95.
  • test substances are contacted with a first protein and a second protein under conditions where the first protein and the second protein bind to each other.
  • the first protein is PSD93, PSD95, or a combination the two proteins.
  • the second protein can be nNOS, NMDA receptor, NR2A subunit, NR2B subunit, or combinations of these proteins.
  • Fusion proteins which contain all or relevant binding portions of these proteins can be used, as is desirable for ease of detectability or purification and handling.
  • the amount of protein which is bound or free in the presence and absence of the test substance can be determined by any techniques known in the art. Test substances which increase the amount of free binding partners or which decrease the amount of bound binding partners are identified as candidate drugs for relieving pain or inducing unconsciousness or sedation.
  • protein-protein binding assays are known in the art and any such format or technique can be used as is convenient.
  • the proteins are contacted in vitro.
  • the proteins are in yeast cells containing recombinant forms of the first and second proteins, and the test substance is contacted with the whole yeast cells.
  • Such assays include the well-known two hybrid assays, in which binding of two binding partners reconstitutes a transcriptional activating activity.
  • the first and second binding partners are each fused to a first and second yeast protein which reconstitute a functional transcriptional activator when brought into physical proximity by binding of the first recombinant protein to the second recombinant protein.
  • Color-metric, enzymatic, or growth assays can be used to determine the transcriptional activation reconstitution. Candidates which are identified as having inhibitory activity in such assays can be further tested in an animal to determine if the candidate drug relieves pain or induces unconsciousness or sedation.
  • PSD-95/SAP90 antisense-treated animals not only experience a significant decrease in MAC for isoflurane, but also experience an attenuation in the NMDA-induced increase in isoflurane MAC.
  • PSD-95/SAP90 appears to mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics. Suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuates responses to painful stimuli mediated through the N-methyl-D-aspartate receptor activation. In spinal cord neurons PSD-95/SAP90 interacts with the N-methyl-D-aspartate receptor subunits 2A 2B.
  • N-methyl-D-aspartate receptor Activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90.
  • PSD-95/SAP90 is required for hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level.
  • PSD-95/SAP90 antisense ODN but not sense or missense ODN produced a remarkable reduction in isoflurane MAC. This was not accompanied by changes in ether blood pressure or heart rate. Furthermore, the PSD-95/SAP90 antisense ODN blocked NMDA-induced increase in isoflurane MAC.
  • the deficiency of PSD- 95/SAP90 expression may produce anesthetic and analgesic actions at the spinal cord level and PSD-95/SAP90 might mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics.
  • Antisense ODNs have been widely used as research tools, and even as drugs in clinical trials. Antisense ODNs inhibit protein expression by the mechanisms of (1) steric blockade of ribosomal subunit attachment to mRNA at the 5' cap site; (2) interference with proper mRNA splicing through antisense binding to splice donor or splice acceptor
  • antisense ODNs only suppressed the expression of PSD-95/SAP90 but not the expression of NMDA receptor subunits NR2A/2B, neuronal nitric oxide synthase or SAP 102 (a protein that is closely related to the targeted protein) in the spinal cord. 49
  • the effects observed following treatment with the PSD- 95/SAP90 antisense ODN are unlikely to be explained by changes in the expression of other proteins.
  • the antisense ODNs at the doses used only affected isoflurane MAC without untoward effects in any of the treated animals including the antisense groups.
  • PSD-95/SAP90 In brain neurons, suppression of PSD-95/SAP90 expression that selectively disrupted physical linkage of the NMDA receptor with neuronal nitric oxide synthase has been demonstrated to attenuate excitotoxicity and Ca2+-activated nitric oxide production via NMDA receptor activity. 23 Mice carrying a targeted mutation in the PSD-95/SAP90 gene showed an enhanced NMDA-dependent long-term potentiation and impaired learning. 16 Recently, we found that PSD-95/SAP90's mRNA and protein also were enriched in the spinal cord and selectively distributed in the superficial dorsal horn, where PSD-95/SAP90
  • PSD-95/SAP90 may be involved in the processing of pain and that deficiency of PSD- 95/SAP90 may produce analgesic action at the spinal cord level. Such an action is consistent with the effect of the deficiency of PSD-95/SAP90 on MAC.
  • Doses of antisense ODNs did not cause motor and general behavioral dysfunction when administered intrathecally in rats.
  • the effect of suppression of spinal PSD-95/SAP90 expression that resulted in the reduction in MAC may be due to effects on analgesia alone.
  • PSD-95/SAP90 has been demonstrated to be involved in the mechanisms of long-term potentiation and learning. 16
  • An effect of antisense ODN on righting reflex was not observed. The possibility of these actions of the antisense ODNs in the central nervous system could not be ruled out from the current study since the intrathecal antisense effect had a segmental nature.
  • PSD-95/SAP90 antibody was able to immunoprecipitate not only PSD-95/SAP90 but also NR2A/2B in vivo. 49 These findings demonstrate that PSD-95/SAP90 interacts with NR2A/2B in the spinal cord in vivo. Combined with the current results, it is suggested that PSD-95/SAP90 is essential for the actions of the NMDA receptor in determining the MAC of inhalational anesthetics.
  • PSD-95/SAP90 MAC for isoflurane was significantly decreased and the NMDA-induced increase in isoflurane MAC was attenuated in the PSD-95/SAP90 antisense-treated animals.
  • Antisense OND given intrathecally at 25 and 50 ⁇ g dramatically prevented the NMDA-induced decrease of the tail-flick latency by 55 % (p ⁇ 0.05) and 82 % (p ⁇ 0.01), respectively.
  • these rats treated with antisense OND were allowed to recover for an additional four days, their tail flick latency in response to NMDA stimulation returned to normal.
  • NMDA-induced thermal hyperalgesia was produced specifically through NMDA receptor activation but not non-NMDA receptor activation, we observed the effects of a selective NMDA receptor antagonist, MK-801, and a selective non-NMDA receptor antagonist, DNQX, on NMDA-induced facilitation of the tail-flick reflex.
  • the baseline thermal reflex is generally considered to be mediated via non-NMDA receptor mechanisms.
  • PSD-95 antisense oligonucleotide acts specifically to inhibit PSD-95 expression.
  • Antisense ONDs widely used as research tools and even as drugs in clinical trials, inhibit protein expression by the mechanisms of (1) steric blockade of ribosomal subunit attachment to mRNA at the 5' cap site; (2) interference with proper mRNA splicing through antisense binding to splice donor or splice acceptor sites; (3) Rnase-H-mediated degradation of hybridized mRNA.
  • PSD-95/SAP90 NMDA receptor subunits 2A/2B (NR2A/2B), neuronal nitric oxide synthase (nNOS) and SAP- 102 in homogenates from crude lumbar enlargement segments in the normal, saline- treated (control) and OND-treated rats.
  • PSD-95/SAP90 protein was enriched in the postsynaptic density (PSD) fraction of the spinal cord in normal, control, sense OND- and missense OND-treated groups (Fig. 1 A a and b).
  • This example demonstrates the expression and localization of PSD95 in the spinal cord, as well as the colocalization with NMDA receptors and nNOS.
  • RNA for messages encoding the PSD- 95/SAP90 protein was extracted from tissues of the spinal cord, other regions of the brain (as positive controls), and muscle (as a negative control). This RNA was probed with the use of RT-PCR analysis.
  • Fig. 2A PSD-95/SAP90 immunoreactivity was found in the spinal cord and distributed mainly in lamina I and outer lamina II. Under high magnification, many PSD-95/SAP90 immunoreactive puncta were observed (Fig. 2B).
  • the superficial dorsal horn not only contains many intemeurons and their processes but also receives the processes from the deep dorsal horn neurons, the primary afferent termini from the periphery and the descending fibers from supraspinal structures. 22 Since PSD-95/SAP90 is specifically localized at synapses and has been found both pre- and post-synaptically in the brain, 3,4 we investigated the sources of PSD-95/SAP90 immunoreactive puncta in the superficial dorsal horn. In the dorsal root ganglion of normal rat, no PSD-95/SAP90 protein was detected (Fig. lAa).
  • PSD-95/SAP90 immunoreactivity in the superficial dorsal horn after unilateral spinal nerve cut or bilateral dorsolateral fasciculi cut (data not shown). More importantly, PSD-95/SAP90 mRNA was detected and PSD-95/SAP90 expression from antisense OND-treated rats was significantly suppressed in the spinal cord as described above. These data indicate that PSD-95/SAP90 in the superficial dorsal horn, to a great extent, is intrinsic to the spinal cord. The superficial dorsal horn is the primary center for processing noxious stimulation.
  • PSD-95/SAP90 has important implications for the mechanisms of nociceptive processing at the spinal cord level.
  • the NMDA receptor has been demonstrated to mainly locate in lamina I and outer lamina II of the spinal cord. 12,2 Combined with the present data, the NMDA receptor completely overlapped with PSD- 95/SAP90 in the spinal dorsal horn. It is suggested that PSD-95/SAP90 may co-localize and interact with the NMDA receptor in the spinal cord neurons.
  • mice Male Sprague-Dawley rats (250-300g) were implanted with an intrathecal PE-10 catheter into the subarachnoid space at the rostral level of the spinal cord lumbar enlargement through an incision at the atlanto- occipital membrane according to the method as described. 25 ' 27 One week or more later, the rats were injected intrathecally with saline or ONDs every 24 h for 4 days.
  • saline, NMDA, MK-801 + NMDA or DNQX + NMDA was given intrathecally.
  • Nociception was evaluated by the radiant heat tail-flick test. The doses and time point of maximal effect of NMDA used in the present study were determined based on a previous study. 24 The tail-flick apparatus (Model 33B Tail Flick Analgesy Meter, IITC Life Science, Woodland Hills, CA, USA) generated a beam of radiant heat that was focused on the underside of the tail, 5 cm from the tip. A cut-off time latency of 13.5 s was used to avoid tissue damage to the tail. Nociception was assessed by the time required to induce tail-flick after applying radiant heat to the skin of the tail.
  • Tail-flick data were expressed as percentage change calculated by the formula: (trial latency - baseline latency) / (baseline latency) x 100 %.
  • PSD1 5'-CAAGCCCAGCAATGCCTA-3'; SEQ ID NO: 3
  • PSD2 5'- CTTGTCGTAATCAAACAG-3'; SEQ ID NO: 4
  • RNA samples (1 ⁇ g) from rat spinal cord, brain and muscle were reverse transcribed to generate first-strand cDNA.
  • the PCR reactions were performed for 25 cycles. Each cycle included 30 s at 94°C, 30 s at 55°C, and 30 s at 71°C.
  • the PCR products were directly cloned into the TA cloning vector (Invitrogen Co., San Diego, CA, USA) and verified by automatic DNA sequencing.
  • PSD fraction was prepared according to procedures described by Luo et al 11 with modifications. In brief, the spinal cord and brain from male Sprague- Dawley rats were homogenized and centrifuged at 800 x g for 10 min to recover the supernatant SI and the pellet PL The SI fraction was subjected to centrifugation at 7,100 x g for 15 min to obtain the pellet P2 and the supernatant S2. P2 was resuspended and again subjected to centrifugation at 8,200 x g for 15 min to recover the synaptosomal fraction P2'.
  • the P2' fraction was treated with an osmotic shock by diluting with double- distilled water and further centrifuged at 25,000 x g for 20 min to generate the pellet LP1 and the supernatant LSI.
  • LP1 was resuspended and centrifuged at 33,000 x g for 20 min.
  • the pellet LP1P was resuspended and loaded onto a discontinuous sucrose gradient composed of 0.10, 1.5 and 2.0 M sucrose. After ultracentrifugation at 208,000 x g for 2 h, the PSD fraction was recovered at the interface between 0.5 and 2.0 M sucrose. The PSD fraction was finally resuspended and centrifuged at 208,000 x g for 30 min.
  • Co-immunoprecipitation and immunoblotting About 2-4 ⁇ g of the affinity-purified mouse PSD-95/SAP90 antibody (Upstate Biotechnology, Lake Placid, NY, USA) was preincubated with 100 ⁇ l of a 1:1 slurry of protein A-sepharose for 1 h, and the protein- antibody complex was spun down at 2,000 rpm for 4 min. The solubilized PSD fraction (400 ⁇ g) was then added to the sepharose beads and the mixture incubated for 2-3 h at 4°C.
  • the mixture was washed once with 1% TritonX-100 in immunoprecipitation buffer (137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 5 mM EGTA, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 50 mM NaF, 20 U/ml Trasylol, and 0.1 mM phenylmethylsulfonyl fluoride), twice with 1% TritonX-100 in immunoprecipitation buffer plus 300 mM NaCl, and three times with immunoprecipitation buffer.
  • immunoprecipitation buffer 137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 5 mM EGTA, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 50 mM NaF, 20
  • the proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane.
  • PSD-95/SAP90 antibody was substituted with normal mouse serum, or was preincubated with excess of PSD-95/SAP90 fusion protein (100 ⁇ g/ml). Immunoblotting was carried out as described by Lau et al 10 .
  • Sections (30 ⁇ m) were cut on a cryostat and then blocked for 1 h in PBS containing 10% goat serum and 0.3% TritonX-100.
  • Primary antibody to PSD-95/SAP90 (1: 1000) was diluted into blocking reagent and incubated with sections overnight. Immunoperoxidase histochemistry was performed using the ABC method. Control sections lacking primary antiserum were stained in parallel.
  • Example 5
  • This example demonstrates the decrease in threshold for isoflurane caused by inhibition of expression of PSD 95.
  • the value for isoflurane MAC in the control (saline-treated) group was 1.16 ⁇ 0.08, which is consistent with that in the previous studies. 49 ' 52 In the groups treated with the antisense ODNs at the doses of 12.5, 25 and 50 ⁇ g, the isoflurane MACs were decreased from isoflurane control MAC of 1%, 18% (P ⁇ 0.01) and 44% (P ⁇ 0.01), respectively (Table 1). In contrast, intrathecal administration of sense ODN at the dose of 50 ⁇ g or missense ODN at the dose of 50 ⁇ g did not significantly change the value for isoflurane MAC compared to the control group (Table 1).
  • antisense ODN reduces the threshold for isolfurane, even in the presence of NMDA which increases the threshold for isoflurane.
  • intrathecal NMDA at a dose of 1.25 ⁇ g caused an increase from isoflurane control MAC by 15% (P ⁇ 0.01; Fig 1).
  • the NMDA-induced change in isoflurane MAC was accompanied by a significant increase in systolic and diastolic blood pressures (135.70 ⁇ 3.38 mmHg and 118.30 ⁇ 7.81 mmHg, respectively.
  • P ⁇ 0.05 vs control but not in heart rate (529.20 ⁇ 55.20 beats/min, P > 0.05 vs control).
  • Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (45 mg/kg). Chronic intrathecal catheters were inserted by passing a polyethylene-10 (PE-10) catheter through an incision in the atlanto-occipital membrane to a position 8 cm caudal to the cisterna at the level of the lumbar subarachinoid space using the methods described previously. The animals were allowed to recover for 5-7 days before experiments were initiated. Rats that showed neurological deficits postoperatively were removed from the study.
  • PE-10 polyethylene-10
  • ODNs were searched to exclude non-specificity of the sense or antisense ODNs and to show that missense ODN did not match any confounding sequences in the GenBank database (GenBank accession number M96853).
  • the ODNs were dissolved in saline before administration.
  • the rats were injected intrathecally with saline (10 ⁇ l) (control), antisense ODNs (12.5, 25, 50 ⁇ g / 10 ⁇ l), sense ODN (50 ⁇ g / 10 ⁇ l) and missense ODN (50 ⁇ g / 10 ⁇ l), respectively, followed by an injection of 10 ⁇ l of saline to flush the catheter, every 24 h for 4 days.
  • each rat On the fifth day after saline or ODNs injection, each rat was placed in a clear plastic cone and anesthetized with 5% isoflurane in oxygen for three to five minutes. After tracheostomy, the trachea of each animal was intubated with a 16-gauge polyethylene catheter. The inspired isoflurane concentration was reduced to 2 %, and the animals breathed spontaneously until cannulation of a carotid artery and a jugular vein with PE-50 tubing was accomplished.
  • a PE-10 catheter was introduced through and beyond the endotracheal tube until obstruction to passage was met and then withdrawn 1 to 2 mm.
  • the PE-10 catheter was connected to a parameter airway gas monitor (Datex-Engstrom, Inc., Tewksbury, MA).
  • MAC was measured according to the methods described previously 34 using a long hemostat (8-inch Rochester Dean Hemostatic Forceps) clamped to the first ratchet lock on the tail for 1 min.
  • the tail was always stimulated proximal to a previous test site. Gross movement of the head, extremities, or body was taken as a positive test result, whereas grimacing, swallowing, chewing, or tail flick were considered negative results.
  • the isoflurane concentration was reduced in decrements of 0.12 to 0.15% until the negative response became positive, with 12-15 min equilibration allowed after changes in concentration. 50 ' 51
  • the MAC was considered to be the concentration midway between the highest concentration that permitted movement in response to the stimulus and the lowest concentration that prevented movement. Finally, intrathecal PE-10 catheter position from each animal was confirmed.
  • NMDA in some saline-treated rats, after initial baseline MAC determination, NMDA at the dose of 1.25 ⁇ g 3 ⁇ or saline was injected intrathecally in a volume of 10 ⁇ l saline, followed by an injection of 10 ⁇ l saline to flush the catheter. Fresh NMDA solution was prepared for each experiment. An isoflurane concentration was chosen at which movement did not occur in the last negative response before the positive test response. At this isoflurane concentration, 10 min after the intrathecal injection of NMDA, the animals were tested again for reactivity to tail clamp. The concentration of isoflurane was increased, and response to tail clamp was checked every 12-15 min thereafter until a negative response was achieved.
  • the effects of ODNs on locomotor function were examined using the following methods. 52
  • the animals were organized randomly into six groups: control (saline); 12.5 ⁇ g antisense ODN; 25 ⁇ g antisense ODN; 50 ⁇ g antisense ODN; 50 ⁇ g sense ODN; 50 ⁇ g missense ODN.
  • the rats were pretreated with ODNs or saline in the manner described above. On the fifth day, 10 ⁇ l of saline was injected intrathecally for each rat. In some saline or antisense ODN (50 ⁇ g)-treated rats, fresh NMDA solution (1.25 ⁇ g / 10 ⁇ l) was injected intrathecally.
  • Placing reflex The rat was held with the hind limbs slightly lower than the forelimbs, and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. The experimenter recorded whether the hind paws were placed on the table surface reflexively; (2) Grasping reflex: The rat was placed on a wire grid and the experimenter recorded whether the hind paws grasped the wire on contact; (3) Righting reflex: The rat was placed on its back on a flat surface and the experimenter noted whether it immediately assumed the normal upright position. Scores for placing, grasping and righting reflexes were based on counts of each normal reflex exhibited in five trials. In addition, the rat general behaviors including spontaneous activity were observed. Statistical analysis
  • the MAC data were assessed statistically by an analysis of variance. Intergroup differences were analyzed using the Newman-Keuls test. Locomotor data were assessed by a rank sum test. All data are reported as the mean ⁇ SD. Significance was set at P ⁇ 0.05.
  • This example demonstrates the role of PSD95 in formalin-induced pain, which is a model for inflammatory-induced pain.
  • Pretreatrnent with PSD-95 antisense ODN produced significant decreases in formalin-induced pain behaviors and c-fos expression in the spinal cord.
  • Intrathecal antisense ODN at 50 ⁇ g reduced the number of flinches and shakes evoked by formalin by 59% (p ⁇ 0.01) in the tonic period but not in the phasic period.
  • the antisense ODN also decreased the number of Fos-like immunoreactive neurons per section by 48% (p ⁇ 0.05).
  • the antisense ODN at 12.5 and 25 ⁇ g failed to produce significant changes in the number of flinches and shakes in the phasic and tonic periods, or in the number of Fos-like immunoreactive neurons, when compared to the saline-treated group.
  • the sense ODN- and the missense ODN-treated groups did not show any significant difference in the number of flinches and shakes in either period, when compared to the saline-treated group.
  • PSD-95 antisense significantly reduced formalin- induced nociceptive behaviors in the tonic period but not in the phasic period. This suggests that PSD-95 protein may play a key role in the spinal sensitization induced by subcutaneous formalin injection.
  • halothane inhibits the NMDA receptor signaling pathway by inhibiting PDZ domain interactions between PSD-95 or PSD-93 and NMDA receptors or nNOS.
  • Both yeast vectors were co-transformed into the Y190 yeast strain, which was then grown in the absence or presence of halothane at clinically relevant concentrations. Protein-protein interactions were confirmed by both yeast growth on -Leu/-Trp/-His (- LTH) medium and lacz expression. To confirm the yeast two-hybrid results, the GST fusion protein binding assay was performed.
  • the GST-fusion proteins consisting of the second PDZ domain of PSD-95 or PSD-93, were expressed in bacterial BL21 cells and purified using glutathione-coupled agarose.
  • GST-PSD-95 or GST-PSD-93 was incubated with membrane proteins from rat hippocampus at room temperature for 1 h. After extensive washing, the bound proteins were eluted by boiling in 1 X SDS-PAGE sample buffer and detected by immunoblotting.
  • This example demonstrates the interaction of PSD-95/SAP90 with NMDA receptor and neuronal nitric oxide synthase (nNOS) were examined.
  • RNA from fissures of the spinal cord, other regions of brain (as positive control) and muscle (as negative control) for messages encoding the PSD- 95/SAP90 protein with the use of RT-PCR analysis.
  • a 0.735 Kb mRNA was detected in the spinal cord and the regions of brain but not in muscle.
  • the PCR product was directly cloned into the TA cloning vector and verified as PSD-95/SAP90 by automatic DNA sequencing.
  • PSD-95/SAP90 protein also was found to enrich in the postsynaptic density fraction of the spinal cord.
  • PSD-95/SAP90 was distributed mainly in spinal superficial laminae, where PSD-95/SAP90 overlapped with NMDA receptor subunits 2A/2B (NR2A/2B) and nNOS, suggesting that PSD- 95/SAP90 might interact with NR2A/2B and nNOS in the spinal cord.
  • endothelial NOS was not immunoprecipitated with PSD-95/SAP90 antibody.
  • the effect of the deficiency of PSD-95/SAP90 on mechanical and thermal hyperalgesia in a rat neuropathic pain model was observed.
  • the antisense oligonucleotide (OND) specifically against PSD-95/SAP90 was employed to reduce the expression of PSD-95/SAP90 in spinal cord.
  • the rats were injected intrathecally with saline (10 ⁇ l), antisense OND (50 ⁇ g/10 ⁇ l) or sense OND (50 ⁇ g/10 ⁇ l) every 24 h for 4 days.
  • the unilateral L5 spinal nerve was ligated. Hind paw withdrawal response to mechanical or heat stimuli was conducted 1 day prior to the surgery and at 3, 5, 7 and 9 days postoperatively.
  • the rat brain postsynaptic density fraction contains a homology of the drosophila discs-large tumor suppressor protein. Neuron 9, 929-942.
  • PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain. J. Biol Chem. 274, 27467-27473.
  • SAP90 a rat presynaptic protein related to the product of the drosophila tumor suppressor gene dig-A. J. Biol. Chem. 268, 4580-4583.
  • Acute thermal hyperalgesia in the rat is produced by activation of N-methyl-D-aspartate receptors and protein kinase C and production of nitric oxide. Neuroscience 71, 327-335.
  • Dickenson AH Sullivan AF: Evidence for a role of the NMDA receptor in the frequency dependent potentiation of deep dorsal horn neurons following C-fiber stimulation. Neuropharmacology 1987; 26: 1235-1238.
  • Kawamata T, Omote K Activation of spinal N-methyl-D-aspartate receptors stimulates a nitric oxide/cyclic guanosine 3,5-monophosphate/glutamate release cascade in nociceptive signaling. nesthesiology 1999; 91 : 1415-1424.
  • Yamamoto T, ShimoyamaN, Mizuguchi T The effect of morphine, MK-801, an NMDA antagonist, and CP-96,345, an NK-1 antagonist, on the hyperalgesia evoked by carrageenan injection in the rat paw.
  • Kennedy MB The postsynaptic density at glutamatergic synapses. Trends Neurosci 1997; 20: 264-268.

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Abstract

Selon l'invention, des animaux traités au moyen de l'antisens PSD-95/SAP90 non seulement voient leur concentration alvéolaire minimum (MAC) en isoflurane baisser de manière significative, mais subissent aussi une atténuation dans l'augmentation induite par le N-méthyl-D-asparate (NMDA) dans la MAC en isoflurane. Il semble que l'antisens PSD-95/SAP90 joue le rôle du récepteur NMDA dans la détermination de la MAC d'anesthésiques par inhalation. Une suppression de l'expression de l'antisens PSD-95/SAP90 dans la moelle épinière atténue de manière significative des réponses à un stimuli douloureux induit par l'activation du récepteur de N-méthyl-D-asparate. Dans la moelle épinière, des neurones PSD-95/SAP90 interagissent avec les sous-unités 2A/2B du récepteur de N-méthyl-D-asparate. Une activation d'un tel récepteur dans l'hyperalgie rachidienne résulte en une association entre le récepteur de N-méthyl-D-asparate et l'antisens PSD-95/SAP90. Ce dernier est nécessaire pour l'hyperalgie déclenchée via le récepteur de N-méthyl-D-asparate au niveau de la moelle épinière.
PCT/US2001/015372 2000-05-12 2001-05-14 Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda Ceased WO2001087285A2 (fr)

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WO2005097090A3 (fr) * 2004-04-05 2006-07-27 Icos Corp Agent interrompant l'interaction psd95 nnos, compositions les contenant, et utilisations therapeutiques associees
WO2008080413A3 (fr) * 2006-12-29 2008-09-12 Tartu Uelikool University Of T Oligonucléotide anti-sens, composition pharmaceutique et procédé pour la régulation négative de l'oxyde nitrique synthase neuronale périphérique et des récepteurs opioïdes mu périphériques, et utilisation d'un oligonucléotide anti-sens pour la prod
CN107312071A (zh) * 2016-04-27 2017-11-03 拜西欧斯（北京）生物技术有限公司 兴奋性神经毒性相关损伤的治疗方法
US20190134150A1 (en) * 2016-04-27 2019-05-09 Biocells (Beijing) Biotech Co., Ltd. Therapeutic methods for excitatory neurotoxicity-related injuries
EP3524257A4 (fr) * 2016-10-10 2020-07-01 Biocells (Beijing) Biotech Co., Ltd. Utilisation d'un polypeptide associé à une lésion nerveuse excitatrice dans la prévention, l'atténuation ou le traitement de la douleur
CN117903259A (zh) * 2023-09-07 2024-04-19 湖南中晟全肽生物科技股份有限公司 一种psd-95抑制剂及其用途

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WO1997033173A1 (fr) * 1996-03-08 1997-09-12 The Regents Of The University Of California Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives
GB9611584D0 (en) * 1996-06-04 1996-08-07 Univ Edinburgh Neurotransmitters
US6103872A (en) * 1998-01-22 2000-08-15 The Johns Hopkins University CAPON: a protein associated with neuronal nitric oxide synthase
CA2273622C (fr) * 1999-06-02 2012-03-20 Michael Tymianski Inhibition de la signalisation induite par le recepteur nmda dans le but de reduire les lesions neuronales

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005097090A3 (fr) * 2004-04-05 2006-07-27 Icos Corp Agent interrompant l'interaction psd95 nnos, compositions les contenant, et utilisations therapeutiques associees
WO2008080413A3 (fr) * 2006-12-29 2008-09-12 Tartu Uelikool University Of T Oligonucléotide anti-sens, composition pharmaceutique et procédé pour la régulation négative de l'oxyde nitrique synthase neuronale périphérique et des récepteurs opioïdes mu périphériques, et utilisation d'un oligonucléotide anti-sens pour la prod
CN107312071A (zh) * 2016-04-27 2017-11-03 拜西欧斯（北京）生物技术有限公司 兴奋性神经毒性相关损伤的治疗方法
US20190134150A1 (en) * 2016-04-27 2019-05-09 Biocells (Beijing) Biotech Co., Ltd. Therapeutic methods for excitatory neurotoxicity-related injuries
EP3524257A4 (fr) * 2016-10-10 2020-07-01 Biocells (Beijing) Biotech Co., Ltd. Utilisation d'un polypeptide associé à une lésion nerveuse excitatrice dans la prévention, l'atténuation ou le traitement de la douleur
CN117903259A (zh) * 2023-09-07 2024-04-19 湖南中晟全肽生物科技股份有限公司 一种psd-95抑制剂及其用途

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