WO2015096654A1 - Shh信号通路特异性抑制剂的应用 - Google Patents

Shh信号通路特异性抑制剂的应用 Download PDF

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WO2015096654A1
WO2015096654A1 PCT/CN2014/094178 CN2014094178W WO2015096654A1 WO 2015096654 A1 WO2015096654 A1 WO 2015096654A1 CN 2014094178 W CN2014094178 W CN 2014094178W WO 2015096654 A1 WO2015096654 A1 WO 2015096654A1
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signaling pathway
shh
shh signaling
epilepsy
downstream
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PCT/CN2014/094178
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French (fr)
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王以政
冯昇杰
马韶蓉
贾彩霞
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

  • the present invention is in the field of biomedicine; more specifically, the invention relates to the use of SHH signaling pathway specific inhibitors.
  • Epilepsy a chronic disease in which sudden abnormal discharge of brain neurons leads to transient brain dysfunction.
  • Epilepsy in China has become the second most common disease in neurology after headache.
  • the etiology of epilepsy is very complex, including genetic factors, abnormal brain development, brain damage and so on.
  • the onset of epilepsy is due to a large accumulation of excitatory neurotransmitter glutamate due to neuronal inhibition and excitatory imbalance.
  • the treatment of epilepsy is mainly drug treatment and surgical removal of the lesion. About 30% of patients with temporal lobe epilepsy are currently insensitive or resistant to existing anti-epileptic drugs.
  • Surgical resection not only has a high risk and high recurrence rate, but may also cause brain function damage (Morimoto K et al. (2004) Kindling and status epilepticus models of epilepsy: rewiring the brain. Progress in neurobiology 73(1): 1-60). Therefore, it has been found that new targets for inducing epilepsy and new drugs are of great significance in the treatment of epilepsy.
  • Sonic Hedgehog is a secreted protein that is both a morphogen and a mitogen and plays a crucial role in the development of the central nervous system (Jiang J & Hui CC (2008) Hedgehog signaling in development and cancer. Developmental Cell 15 (6): 801-812).
  • an SHH signaling pathway inhibitor for the preparation of a composition for ameliorating or treating epilepsy.
  • the ameliorating or treating epilepsy comprises:
  • the SHH signaling pathway inhibitor comprises: Cyclopamine, GDC-0449, or an analog thereof.
  • a SHH signaling pathway for screening for potential substances that improve or treat epilepsy; preferably, the screening does not include methods associated with disease diagnosis or treatment methods.
  • a method of screening for a potential substance that ameliorates or treats epilepsy comprising:
  • the candidate substance can reduce the expression amount, activity or secretion amount of the SHH protein, or decrease the phosphorylation level of the molecule downstream of the SHH signaling pathway or the expression level of the transcription factor downstream of the SHH signaling pathway, it indicates that the candidate substance is improved or A potential substance for the treatment of epilepsy.
  • step (1) comprises: adding a candidate substance to the system comprising the SHH signaling pathway in the test set; and/or
  • Step (2) comprises: detecting the expression level, activity or secretion amount of SHH protein in the test group system, or detecting the phosphorylation level of the molecule downstream of the SHH signaling pathway or the expression level of the transcription factor downstream of the SHH signaling pathway, and comparing with the control group Wherein the control group is a system comprising the SHH signaling pathway without adding the candidate substance;
  • the amount, activity or secretion of SHH protein in the test group is statistically lower (preferably significantly lower than, for example, lower than 20%, preferably lower than 50%; more preferably lower than 80%) , or the phosphorylation level of the molecule downstream of the SHH signaling pathway or the expression level of the transcription factor downstream of the SHH signaling pathway is statistically lower (preferably significantly lower than, for example, lower than 20%, preferably lower than 50%; more preferably A control group of less than 80% lower indicates that the candidate is a potential substance for improving or treating epilepsy.
  • the system is selected from the group consisting of a cellular system (such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present), a subcellular system, a solution system, a tissue system, an organ system, or an animal system. .
  • a cellular system such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present
  • a subcellular system such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present
  • a subcellular system such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present
  • a subcellular system such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present
  • a subcellular system such as a hippocampal neuron or a cell culture in which an SHH signaling pathway is endogenously present
  • a subcellular system
  • system is a cellular system.
  • the system is a hippocampal neuron or a cell culture thereof endogenously presenting a SHH signaling pathway.
  • the candidate substance includes, but is not limited to: for the presence of the SHH signal A small molecule compound designed by a pathway (a gene or protein containing the pathway), an interference molecule designed against the SHH signaling pathway or an upstream or downstream protein thereof, a nucleic acid inhibitor, a binding molecule (such as an antibody or a ligand), a small molecule compound, and the like.
  • the method further comprises performing further cellular experiments and/or animal tests on the obtained potential substances to further select and determine substances useful for improving or treating epilepsy from the candidate substances.
  • composition for ameliorating or treating epilepsy comprising:
  • SHH increases extracellular glutamate levels by inhibiting glutamate transporters.
  • AC the change in extracellular glutamate levels was measured by high performance liquid chromatography. Changes in glutamate levels in hippocampal neuronal cultures were measured after administration of a blank control, SHH, Cyclopamine (Cyclo) or SHH and Cyclo stimulation. Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. *p ⁇ 0.05;**p ⁇ 0.01;***p ⁇ 0.001 vs. control group.
  • DF 3 H glutamate uptake test, the change of glutamate uptake ability of cultured hippocampal neurons after stimulation with blank control, SHH, Cyclo or SHH and Cyclo. Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. ***p ⁇ 0.001 vs. control group.
  • the secretion level of SHH in primary cultured hippocampal neurons was measured under conditions simulating epilepsy stimulation.
  • A hippocampal neurons were incubated with normal external fluid and without Mg 2+ external solution (0Mg 2+ ) for 15 minutes, 30 minutes or 60 minutes, and the secretion of SHH in the extracellular fluid was detected by ELISA.
  • B hippocampal neurons were incubated with normal external solution and an external solution containing 100 ⁇ M PTX, 6.5 mM KCl (K + ) for 15 minutes, 30 minutes or 60 minutes, and the amount of SHH secreted in the extracellular fluid was measured by ELISA. Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. *p ⁇ 0.05;**p ⁇ 0.01 vs. control group.
  • the epileptic discharge of the neurons after stimulation was recorded on primary cultured hippocampal neurons by whole-cell recording.
  • A incubate with normal external fluid or without Mg 2+ external solution, record neuronal epileptiform discharge in the presence of Cyclo (concentration 10 ⁇ M) or vehicle, and count the discharge frequency.
  • B incubate with normal external fluid or PTX, 6.5 mM KCl external solution, record neuronal epileptiform discharge in the presence of Cyclo (concentration 10 ⁇ M) or vehicle, and count the discharge frequency.
  • Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. **p ⁇ 0.01;***p ⁇ 0.001 vs. control group.
  • C PTX, 6.5 mM KCl external solution, in the presence of GDC-0449 (concentration 10 ⁇ M) or vehicle, an example of neuronal epileptiform discharge.
  • Cyclopamine inhibits the frequency of neuronal epileptiform discharges in hippocampal slices.
  • A hippocampal slices were incubated with normal external solution and an external solution containing 100 ⁇ M PTX, 6.5 mM KCl for 30 minutes, and after 1 hour or 1.5 hours, the secretion of SHH in the extracellular fluid was measured by ELISA. Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. *p ⁇ 0.05 vs. control group. The epileptic discharge of the neurons after stimulation was recorded on the hippocampal slices by the method of field potential recording. B, representative population peak potential plots recorded in the presence or absence of Cyclo incubation (concentration 10 ⁇ M); 1, Control Condition 2, Cyclo Incubation Conditions. C, group peak potential amplitude statistics. Data were collected from three or more independent experiments and expressed as mean ⁇ standard error. *p ⁇ 0.05 vs. control group.
  • Cyclopamine inhibits seizures in a mouse electrical stimulation igniting model.
  • A model diagram of the method of establishing a mouse electrical stimulation ignition model. Mice were injected intraperitoneally with control solvent or Cyclopamine (10 mg/kg) to determine the effect of Cyclo on the ignition process in mice. B, mouse seizure grade. C, the number of stimuli required for the mouse to reach full ignition. Data are expressed as mean ⁇ standard error from 28 mice, of which 14 were in the control group and the drug-administered group, respectively. *p ⁇ 0.05**p ⁇ 0.01; ***p ⁇ 0.001 vs. control group.
  • Cyclopamine inhibits the formation of spontaneous epilepsy in a mouse model of pilocarpine.
  • A the frequency of spontaneous seizures per mouse per week.
  • B the frequency of grade 4-5 spontaneous seizures per mouse per week.
  • C the percentage of grade 5 spontaneous epilepsy in the two groups of mice. Data are expressed as mean ⁇ standard error from 45 mice, of which 22 were in the control group and 23 in the drug-administered group. **p ⁇ 0.01; ***p ⁇ 0.001 vs. control group.
  • the inventors revealed for the first time that the SHH signaling pathway regulates the excitability of neural networks and participates in the formation and development of epilepsy, and thus can be used as a drug target for developing drugs for improving or treating epilepsy.
  • specific inhibitors of the SHH signaling pathway such as Cyclopamine and GDC-0449
  • the present invention provides the use of an inhibitor of the SHH signaling pathway for the preparation of a composition for ameliorating or treating epilepsy.
  • inhibitors of the SHH signaling pathway gene or protein include antagonists, down-regulators, blockers, blockers, and the like.
  • the SHH signaling pathway gene or protein inhibitor refers to any activity which can reduce the activity of the SHH signaling pathway protein, decrease the stability of the SHH signaling pathway gene or protein, down-regulate the expression of the SHH signaling pathway protein, and reduce the effective effect of the SHH signaling pathway protein.
  • the inhibitor may be: a small interfering RNA molecule or an antisense nucleotide that specifically interferes with expression of the SHH signaling pathway gene; or an antibody or ligand that specifically binds to the SHH signaling pathway protein.
  • the SHH signaling pathway inhibitor comprises: Cyclopamine, GDC-0449, or an analog thereof.
  • the present invention also provides a composition
  • a composition comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the inhibitor of the SHH signaling pathway, and A pharmaceutically acceptable carrier.
  • the composition can be used to ameliorate or treat epilepsy. Any of the foregoing inhibitors of the SHH signaling pathway can be used in the preparation of the composition.
  • phrases “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • the “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential in themselves. Active ingredient, and not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, or a buffer.
  • auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the inhibitor can be administered to a mammal using a variety of methods well known in the art. These include, but are not limited to, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the administration mode is parenterally administered.
  • the effective amount of the inhibitor of the SHH signaling pathway of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the inhibitor of the SHH signaling pathway gene or protein, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, The patient's immune status, route of administration, etc.
  • the inhibitor of the SHH signaling pathway of the present invention when administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the present invention provides a method of screening for a potential substance for ameliorating or treating epilepsy, the method comprising: treating a system comprising (expressing) a SHH signaling pathway with a candidate substance; and detecting expression of a SHH signaling pathway protein in the system Amount or activity; if the candidate substance can down-regulate the expression level or activity of the SHH signaling pathway protein (including the SHH protein), it indicates that the candidate substance is a potential substance for improving or treating epilepsy.
  • the system comprising the SHH signaling pathway may be, for example, a cell (or cell culture) system, and the cells may be cells endogenously containing (expressing) the SHH signaling pathway; or may be cells recombinantly expressing the SHH signaling pathway. .
  • the system comprising the SHH signaling pathway may also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal such as a mouse, a rabbit, a sheep, a monkey, etc. )Wait.
  • the SHH signaling pathway egg is more easily observed during screening.
  • a control group may also be provided for the change in expression or activity of white, and the control group may be a system comprising the SHH signaling pathway without adding the candidate substance.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for improving or treating epilepsy.
  • the method for detecting the expression, activity, expression amount or secretion of the SHH signaling pathway protein is not particularly limited.
  • Conventional protein quantification or semi-quantitative detection techniques can be employed, such as, but not limited to, SDS-PAGE, Western-Blot, and the like.
  • the invention also provides a potential substance for improving or treating epilepsy obtained by the screening method.
  • These initially screened materials can constitute a screening library so that one can ultimately screen for substances that are useful for inhibiting the expression and activity of the SHH signaling pathway, thereby improving or treating epilepsy.
  • the SHH signaling pathway is closely related to the occurrence or development of epilepsy, and has nothing to do with the function of morphogens and mitogens, so that it can be used as a drug target to develop drugs for improving or treating epilepsy.
  • the cells were washed twice with the extracellular fluid, and the external solution containing BSA and the external solution containing SHH were incubated for 30 minutes each. The two incubation solutions were separately collected, and the perchloric acid precipitated protein was added, and the supernatant was left for high performance liquid chromatography analysis.
  • a high performance liquid chromatography system was used: Agilent 1200 series HPLC-FLD, A CAPCELL PAK C183.0 mm I.D x 75 mm analytical column.
  • the excitation wavelength was 340 nm and the emission wavelength was 450 nm.
  • the column temperature was controlled at 36 °C.
  • the mobile phase consists of methanol, acetonitrile and water.
  • the cells were washed twice with extracellular fluid (6 mM glucose, 4 mM KCl, 130 mM NaCl, 1.3 mM CaCl 2 , 1.2 mM MgSO 4 , 1 mM KH 2 PO 4 , 25 HEPES [pH 7.3]) for 10 minutes each time, containing SHH or The extracellular fluid of the inhibitor was preincubated for 10 minutes, and L-type glutamate labeled with a final concentration of 10 -6 M ⁇ was added, the activity was 25 Ci/mmol, and incubation was carried out for 6 minutes at 37 °C. At the end of the reaction, 1 ml of extracellular solution at 4 ° C was quickly added and the cells were placed on ice.
  • extracellular fluid 6 mM glucose, 4 mM KCl, 130 mM NaCl, 1.3 mM CaCl 2 , 1.2 mM MgSO 4 , 1 mM KH 2 PO 4 , 25 HEPES [pH
  • the cells were washed twice with a pre-cooled extracellular solution, and the cells were lysed by adding 1 equivalent of NaOH, and the supernatant was taken at 10,000 g for 5 minutes, and the radioactivity was recorded by a liquid scintillation apparatus.
  • the amount of SHH secreted was detected by enzyme-linked immunosorbent assay kit (R&D MSHH00).
  • the linear range of the standard curve is 0-500 pg/ml, and the content of SHH is within this range.
  • Electrophysiological recordings were performed after incubation for 1 h in artificial cerebrospinal fluid.
  • the stimulating electrode is located in the CA1 radiation layer, and the recording electrode is located in the cell layer of CA1.
  • the distance between the two electrodes is about 300 ⁇ m.
  • the stimulation was given every 20 s and the stimulation wave width was 0.1 ms.
  • the stimulation intensity is the stimulation intensity at 30% to 40% of the maximum response amplitude.
  • the administration was perfused after the reaction amplitude was stabilized for 15 minutes.
  • Hippocampal neurons cultured in vitro for about 10 days were cultured in the external solution for 30 minutes and then subjected to whole-cell patch clamp recording.
  • the electrode liquid is a potassium gluconate solution.
  • the zero magnesium external liquid removes magnesium ions from the normal external liquid.
  • the PTX concentration was 100 ⁇ M and the KCl was 6.5 mM.
  • mice were anesthetized and fixed with a stereotactic locator.
  • the left amygdala (P: -0.12, L: +0.28, D: -0.46) was positioned using the anterior iliac crest and buried in the electrode.
  • the right skull is screwed into the screw, one of which is wound around the ground wire. After fixing with dental cement, it was recovered for 7 days, and threshold measurement was performed.
  • the stimulation frequency is 60 Hz
  • the wave width is 1 ms
  • the stimulation time is 1 s.
  • Mice were stimulated daily with threshold intensity and the length of behavior and post-potentials were recorded until the animals were fully ignited (3 occurrences of 5 and defined as complete igniting).
  • the epileptic behavioral index refers to the Racine classification method (1972): level 0, no change in behavior in mice; level 1, facial twitching, whisker shaking; level 2, involuntary nodding and regular blinking; level 3, unilateral forelimb stiffness and Lift up; level 4, raised both forelimbs, upright; level 5, The hind limbs are lifted up, erect and squat, losing posture control.
  • mice C57BL/6 adult male mice were injected with 2 mg/kg of scopolamine methyl nitrate (Sigma) to block the side effects of pilocarpine. After 30 minutes, the dose of 300 mg/kg of pilocarpine was injected. Pilocarpine hydrochloride (Sigma) was dissolved in 0.9% (w/v) saline, and the time from the first episode to grade 5 epilepsy was recorded. When the mice maintained persistent epilepticus for 2 hours, they were injected intraperitoneally. The dose was 4 mg/kg of Diazepam (Sigma).
  • the present inventors used high performance liquid chromatography (HPLC) to detect extracellular glutamate in cultured hippocampal neurons (isolated rat hippocampal primary culture).
  • HPLC high performance liquid chromatography
  • SHH purchased from Sigma
  • Cyclopamine cyclopamine, purchased from Sigma
  • Fig. 1A where SHH concentration was 500 ng/ml
  • Cyclopamine 10 ⁇ M an inhibitor of this signaling pathway
  • the inventors used the method of 3 H-labeled glutamic acid to observe whether SHH affects the uptake of glutamate.
  • the experimental results show that SHH is similar to the glutamate transporter inhibitor TBOA, which can significantly inhibit the uptake of glutamate by the cells, and this effect is also SHH concentration dependent (Fig. 1D-F; SHH concentration in D 500ng /ml, Cyclopamine concentration 10 ⁇ M; F, DHK concentration 300 ⁇ M SHH concentration 500 ng/ml, TBOA concentration 100 ⁇ M).
  • the method of the present invention for whole-cell patch clamp recording is to observe the discharge of neurons after pre-incubation of no external solution of Mg 2+ or PTX and high concentration of KCl for 30 minutes, and it is found that both of the stimuli can be in vitro.
  • the neurons were induced to spontaneous epileptic discharge, and there was almost no such abnormal discharge in the normal external fluid. And under these two conditions, the frequency of induced epileptic discharges can be inhibited by Cyclopamine (Fig. 3A, B).
  • GDC-0449 also inhibited PTX-induced epileptic discharge frequency (Fig. 3C).
  • Cyclopamine can reduce the amplitude of the peak potential of hippocampal neurons in acute brain slices of rats
  • the present inventors used a method of electrophysiological recording of acute excised brain slices in adult rats.
  • Picrotoxin 100 ⁇ m was added to the artificial cerebrospinal fluid of the brain slices, and the potassium ion concentration (6.5 mM) was increased to induce seizures.
  • the ELISA results showed a large release of SHH.
  • the inventors recorded the stimulation-induced multi-level group peak potential extracellularly in the hippocampal CA1 cell layer. After Cyclopamine was perfused, the amplitudes of the group peaks decreased. The amplitude of the first peak potential was counted and there was a significant difference after 15 minutes of perfusion (Fig. 4).
  • Vismodegib (GDC-0449) (purchased from Selleck) is another specific inhibitor of the SHH pathway directed against Smo. Due to its fewer side effects and good therapeutic effects, GDC-0449 was approved by the US FDA for the treatment of basal cell tumors on January 30, 2012. As of June 2011, the drug is still undergoing treatment with a variety of cancers, including gastric cancer, pancreatic cancer, medulloblastoma, small cell lung cancer, and chondrocytes. Tumor, colon cancer, clinical trial of treatment.
  • GDC-0449 In order to further verify the role of the SHH pathway in epileptic conditions and prepare for subsequent clinical trials, the inventors examined the effects of GDC-0449 in hippocampal neurons cultured in vitro and in mouse electrical stimulation igniting. It was found that GDC-0449 has similar effects to Cyclopamine, which can also delay epileptogenesis and inhibit epileptic discharge.
  • Hippocampal neuronal cells are obtained, which express related molecules of the SHH signaling pathway. This cell was used as a cell model for screening for drugs that inhibit the SHH signaling pathway.
  • Test group a culture of the above cells treated with a candidate substance
  • Control group A culture of the above cells treated without a candidate substance.
  • the activation of the SHH signaling pathway in the cells was determined by conventional methods at appropriate times after treatment.
  • References Jin Jiang, et al. (2008) Hedgehog Signaling in Development and Cancer, Developmental Cell 15: 801-812 (described in the SHH signaling pathway, phosphorylation levels of downstream molecules, such as phosphorylation of Smo, etc., or The level of expression of downstream transcription factors such as Gli1, Gli2, etc., can specifically indicate the activation of this pathway), if compared with the control group, the phosphorylation level of Smo or the mRNA or protein expression of Gli1/Gli2 in the test group
  • a significant drop of more than 20% indicates that the candidate substance is a potential substance for the treatment of epilepsy.
  • Cyclopamine and GDC-0449 were used as candidate substances. The results showed that the mRNA and protein levels of Gli1 and Gli2 were significantly decreased, suggesting that the activity of SHH signaling pathway was significantly down-regulated, and Cyclopamine and GDC-0449 were potential treatments. The substance of epilepsy.
  • Hippocampal neuronal cells are obtained, which express related molecules of the SHH signaling pathway. This cell was used as a cell model for screening for drugs that inhibit the SHH signaling pathway.
  • Test group a culture of the above cells treated with a candidate substance
  • Control group A culture of the above cells treated without a candidate substance.
  • the amount, activity or amount of SHH protein expression of the cells is determined by a conventional method at an appropriate time after the treatment. If the amount, activity or secretion of SHH protein in the test group is significantly decreased compared with the control group, it indicates that the candidate substance is a potential substance for treating epilepsy.
  • Example 7 cyclopamine inhibits the formation of spontaneous epilepsy in an animal model
  • mice were randomly divided into two groups, one of which served as a blank control for intraperitoneal injection of cyclopamine in two days: 45% HBC, and the other group as an experimental group for intraperitoneal injection of 10 mg/kg every two days. Cyclopamine.
  • the video was combined with the age and frequency of spontaneous seizures in mice at 4, 6, and 8 weeks after manual recording. Each mouse was observed for 7 hours per day for 15 days. The epileptic behavioral grading and mouse electrical stimulation were ignited.
  • the model is the same.
  • the results of the experiment showed that the frequency of spontaneous epileptic seizures decreased in all grades compared with the control group, and the frequency of severe spontaneous epilepsy (4-5) occurred. The decline is more significant.
  • the proportion of mice with at least one episode of epilepsy at least during the observation period was counted, and the control group was approximately three times as large as the administration group, as shown in Fig. 6.

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Abstract

本发明涉及SHH信号通路特异性抑制剂的应用。首次揭示了SHH信号通路和神经网络兴奋性有着密切的关系，参与了癫痫的形成与发展，从而可以作为药物靶点，用于开发改善或治疗癫痫的药物。还揭示了可起到延缓癫痫发生的SHH信号通路的特异性抑制剂，可以作为改善或治疗癫痫的药物。

Description

SHH信号通路特异性抑制剂的应用 技术领域
本发明属于生物医药领域；更具体地，本发明涉及SHH信号通路特异性抑制剂的应用。
背景技术
癫痫(epilepsy)，是大脑神经元突发性异常放电，导致短暂的大脑功能障碍的一种慢性疾病。世界范围内，约有1％人口患有癫痫。在中国癫痫已经成为神经科仅次于头痛的第二大常见病。癫痫的病因十分复杂，包括遗传因素，大脑发育异常，脑损伤等等。总体来说，癫痫的发病起因是由于神经网络抑制性和兴奋性失衡，而导致的兴奋型神经递质谷氨酸的大量堆积。癫痫的治疗方法主要是药物治疗和手术切除病灶。目前约有30％的颞叶癫痫患者对现有的抗癫痫药物不敏感或是产生耐药。而手术切除不但高风险高复发率，且可能导致大脑功能损伤(Morimoto K等(2004)Kindling and status epilepticus models of epilepsy:rewiring the brain.Progress in neurobiology 73(1):1-60)。因此，发现诱导癫痫的新靶点及新药物对抗癫痫的治疗具有重要的意义。
Sonic Hedgehog(SHH)是一种分泌蛋白，它既是形态发生素又是促分裂素，在中枢神经系统发育中起着至关重要的作用(Jiang J&Hui CC(2008)Hedgehog signaling in development and cancer.Developmental cell 15(6):801-812)。
然而，虽然SHH信号通路在成年动物的大脑中依然有着广泛的表达，但其作用却不完全清楚。
发明内容
本发明的目的在于提供SHH信号通路特异性抑制剂在改善或治疗癫痫方面的应用。
在本发明的第一方面，提供一种SHH信号通路抑制剂的用途，用于制备改善或治疗癫痫的组合物。
在一个优选例中，所述改善或治疗癫痫包括：
延缓癫痫发生；
抑制海马神经元癫痫样放电；和/或
降低癫痫发作强度。
在另一优选例中，所述的SHH信号通路抑制剂包括：环巴胺(Cyclopamine)，GDC-0449，或它们的类似物。
在本发明的另一方面，提供SHH信号通路的用途，用于筛选改善或治疗癫痫的潜在物质；较佳地，所述筛选不包括疾病诊断或治疗方法相关的方法。
在本发明的另一方面，提供一种筛选改善或治疗癫痫的潜在物质的方法，所述方法包括：
(1)用候选物质处理包含SHH信号通路的体系；
(2)检测所述体系中SHH蛋白的表达量、活性或分泌量，或检测SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量；
其中，若所述候选物质可降低SHH蛋白的表达量、活性或分泌量，或降低SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量，则表明该候选物质是改善或治疗癫痫的潜在物质。
在一个优选例中，步骤(1)包括：在测试组中，将候选物质加入到包含SHH信号通路的体系中；和/或
步骤(2)包括：检测测试组的体系中SHH蛋白的表达量、活性或分泌量，或检测SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量，并与对照组比较，其中所述的对照组是不添加所述候选物质的包含SHH信号通路的体系；
如果测试组中SHH蛋白的表达量、活性或分泌量在统计学上低于(优选显著低于，如低20％以上，较佳的低50％以上；更佳的低80％以上)对照组，或SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量在统计学上低于(优选显著低于，如低20％以上，较佳的低50％以上；更佳的低80％以上)对照组，就表明该候选物是改善或治疗癫痫的潜在物质。
在另一优选例中，所述的体系选自：细胞体系(如内源存在SHH信号通路的海马神经元或其细胞培养物)、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
在另一优选例中，所述的体系是细胞体系。
在另一优选例中，所述的体系是内源存在SHH信号通路的海马神经元或其细胞培养物。
在另一优选例中，所述的候选物质包括(但不限于)：针对所述存在SHH信号 通路(含该通路的基因或蛋白)设计的小分子化合物，针对SHH信号通路或其上游或下游蛋白设计的干扰分子、核酸抑制物、结合分子(如抗体或配体)、小分子化合物等。
在另一优选例中，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以从候选物质中进一步选择和确定对于改善或治疗癫痫有用的物质。
在本发明的另一方面，提供一种用于改善或治疗癫痫的组合物，所述的组合物含有：
(1)环巴胺(Cyclopamine)，GDC-0449，或它们的类似物；和
(2)药学上可接受的载体。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、SHH通过抑制谷氨酸转运体增加胞外谷氨酸水平。
(A-C)，用高效液相色谱的方法检测胞外谷氨酸水平的变化。检测在给予空白对照，SHH，Cyclopamine(Cyclo)或是SHH和Cyclo刺激后，海马神经元培养液中谷氨酸水平的变化。数据从三组以上独立的实验收集，用平均值±标准误表示。*p<0.05；**p<0.01；***p<0.001相对于对照组。(D-F)，3H谷氨酸摄取实验，检测给与空白对照，SHH，Cyclo或是SHH和Cyclo刺激后，培养的海马神经元谷氨酸摄取能力的变化。数据从三组以上独立的实验收集，用平均值±标准误表示。***p<0.001相对于对照组。
图2、癫痫性刺激促进SHH的分泌。
在模拟癫痫刺激的条件下检测原代培养的海马神经元SHH的分泌水平。A，分别给予正常外液和无Mg2+外液(0Mg2+)孵育海马神经元15分钟，30分钟或60分钟后，用ELISA的方法检测细胞外液中SHH的分泌量。B，分别给予正常外液和含有100μM PTX，6.5mM KCl(K+)的外液孵育海马神经元15分钟，30分钟或60分钟后，用ELISA的方法检测细胞外液中SHH的分泌量。数据从三组以上独立的实验收集，用平均值±标准误表示。*p<0.05；**p<0.01相对于对照组。
图3，Cyclopamine和GDC-0449抑制神经元癫痫样放电频率。
用全细胞记录的方法，在原代培养的海马神经元上记录给与刺激后神经元癫痫样放电的情况。A，用正常外液或无Mg2+外液孵育，在存在Cyclo(浓度10μM)或是vehicle的条件下，记录神经元癫痫样放电，统计放电频率。B，用正常外液或PTX，6.5mM KCl外液孵育，在存在Cyclo(浓度10μM)或是vehicle的条件下，记录神经元癫痫样放电，统计放电频率。数据从三组以上独立的实验收集，用平均值±标准误表示。**p<0.01；***p<0.001相对于对照组。C，PTX，6.5mM KCl外液孵育，在存在GDC-0449(浓度10μM)或是vehicle的条件下，神经元癫痫样放电示例图。
图4，Cyclopamine抑制海马脑片中神经元癫痫样放电频率。
A，分别给与正常外液和含有100μM PTX，6.5mM KCl的外液孵育海马脑片30分钟，1小时或1.5小时后，用ELISA的方法检测细胞外液中SHH的分泌量。数据从三组以上独立的实验收集，用平均值±标准误表示。*p<0.05相对于对照组。用场电位记录的方法，在海马脑片上记录给与刺激后神经元癫痫样放电的情况。B，在存在或是不存在Cyclo孵育(浓度10μM)的条件下，记录的代表性群峰电位图；1，对照条件2，Cyclo孵育条件。C，群峰电位幅度统计图。数据从三组以上独立的实验收集，用平均值±标准误表示。*p<0.05相对于对照组。
图5，在小鼠电刺激点燃模型中，Cyclopamine抑制癫痫发作。
A，小鼠电刺激点燃模型建立方法模式图。给小鼠腹腔注射对照溶剂或Cyclopamine(用量10mg/kg)，检测Cyclo对小鼠点燃进程的影响。B，小鼠癫痫发作等级。C，小鼠达到完全点燃所需的刺激数。数据用平均值±标准误表示，来自于28只小鼠，其中对照组与给药组分别为14只。*p<0.05**p<0.01；***p<0.001相对于对照组。
图6，在小鼠匹鲁卡品的模型中，Cyclopamine抑制自发性癫痫的形成。
A，每周每只鼠自发性癫痫的发作频率。B，每周平均每只小鼠发4-5级自发性癫痫的频率。C，两组小鼠发5级自发性癫痫的百分比。数据用平均值±标准误表示，来自于45只小鼠，其中对照组为22只，给药组为23只。**p<0.01；***p<0.001相对于对照组。
具体实施方式
本发明人经过系统和深入的研究，首次揭示了SHH信号通路调节神经网络的兴奋性，并参与癫痫的形成与发展，从而可以作为药物靶点，用于开发改善或治疗癫痫的药物。本发明人还发现，SHH信号通路的特异性抑制剂(如Cyclopamine和GDC-0449)可起到延缓癫痫发生，抑制癫痫样放电的作用，可以作为改善或治疗癫痫的药物。
SHH信号通路抑制剂及其用途
基于本发明人的上述新发现，本发明提供了一种SHH信号通路的抑制剂的用途，用于制备改善或治疗癫痫的组合物。
如本文所用，所述的SHH信号通路基因或蛋白的抑制剂包括了拮抗剂、下调剂、阻滞剂、阻断剂等。
所述的SHH信号通路基因或蛋白的抑制剂是指任何可降低SHH信号通路蛋白的活性、降低SHH信号通路基因或蛋白的稳定性、下调SHH信号通路蛋白的表达、减少SHH信号通路蛋白有效作用时间、或抑制SHH信号通路基因的转录和翻译的物质，这些物质均可用于本发明，作为对于下调SHH信号通路有用的物质，从而可用于改善或治疗癫痫。例如，所述的抑制剂可以是：特异性干扰SHH信号通路基因表达的小干扰RNA分子或反义核苷酸；或特异性与SHH信号通路蛋白结合的抗体或配体。
作为本发明的优选方式，所述的SHH信号通路抑制剂包括：Cyclopamine，GDC-0449，或它们的类似物。
组合物
本发明还提供了一种组合物，它含有有效量(如0.000001-50wt％；较佳的0.00001-20wt％；更佳的，0.0001-10wt％)的所述的SHH信号通路的抑制剂，以及药学上可接受的载体。所述的组合物可用于改善或治疗癫痫。任何前述的SHH信号通路的抑制剂均可用于组合物的制备。
如本文所用，所述“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。所述“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的 活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。
在得知了所述SHH信号通路的抑制剂的用途后，可以采用本领域熟知的多种方法来将所述的抑制剂、或其药物组合物给药于哺乳动物。包括但不限于：皮下注射、肌肉注射、经皮给予、局部给予、植入、缓释给予等；优选的，所述给药方式是非肠道给予的。
本发明所述的SHH信号通路的抑制剂的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的SHH信号通路基因或蛋白的抑制剂的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的SHH信号通路的抑制剂每天以约0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
药物筛选
在得知了SHH信号通路与癫痫的密切相关性后，可以基于该特征来筛选抑制SHH信号通路的物质。可从所述的物质中找到对于改善或治疗癫痫真正有用的药物。
因此，本发明提供一种筛选改善或治疗癫痫的潜在物质的方法，所述的方法包括：用候选物质处理包含(表达)SHH信号通路的体系；和检测所述体系中SHH信号通路蛋白的表达量或活性；若所述候选物质可下调SHH信号通路蛋白(包括SHH蛋白)的表达量或活性，则表明该候选物质是改善或治疗癫痫的潜在物质。所述的包含SHH信号通路的体系例如可以是细胞(或细胞培养物)体系，所述的细胞可以是内源性包含(表达)SHH信号通路的细胞；或可以是重组表达SHH信号通路的细胞。所述的包含SHH信号通路的体系还可以是亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。
在本发明的优选方式中，在进行筛选时，为了更易于观察到SHH信号通路蛋 白的表达或活性的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的包含SHH信号通路的体系。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于改善或治疗癫痫真正有用的物质。
本发明对于SHH信号通路蛋白的表达、活性、表达量或分泌情况的检测方法没有特别的限制。可以采用常规的蛋白定量或半定量检测技术，例如(但不限于)：SDS-PAGE法，Western-Blot法等。
另一方面，本发明还提供了采用所述筛选方法获得的改善或治疗癫痫的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于抑制SHH信号通路的表达和活性，进而改善或治疗癫痫有用的物质。
本发明的主要优点在于：
首次揭示了SHH信号通路与癫痫的发生或发展密切相关，并且与其形态发生素和促分裂素的功能无关，从而可以作为药物靶点开发改善或治疗癫痫的药物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料与方法
1、高效液相色谱分析
细胞用胞外液洗两次，先后孵育含BSA外液及含SHH外液各30分钟。分别收集此两种孵育液，加入高氯酸沉淀蛋白，上清留作高效液相色谱分析。高效液相色谱系统采用：Agilent 1200series HPLC-FLD，A CAPCELL PAK C183.0mmI.D×75mm分析柱。激发波长为340nm，发射波长为450nm。柱温控制在36℃。流动相由甲醇，乙腈和水组成。
2、氚标记谷氨酸摄取实验
细胞用胞外液(6mM葡萄糖，4mM KCl，130mM NaCl，1.3mM CaCl2，1.2 mM MgSO4，1mM KH2PO4，25HEPES[pH 7.3])洗涤两次，每次10分钟，用含有SHH或抑制剂的胞外液预孵10分钟，加入终浓度为10-6M氚标记的L型谷氨酸，活性为25Ci/mmol，37℃孵育6分钟。反应结束时迅速加入1ml 4℃胞外液，细胞冰上放置。用预冷胞外液洗两次，加入1当量的NaOH裂解细胞，10000g 5分钟取上清，用液闪仪记录放射活性。
3、酶联免疫吸附实验
SHH的分泌量用酶联免疫吸附试剂盒(R&D MSHH00)检测。标准曲线的线性范围是0-500pg/ml，SHH的含量均在此范围内得出。
4、大鼠急性脑片电生理记录
取120～180gSD大鼠深度麻醉后取脑，用振动切片机(LeciaVT1000s)切成350μm厚的海马脑片。在人工脑脊液中孵育1h后进行电生理记录。刺激电极位于CA1辐射层，记录电极位于CA1的胞体层。两电极间距离约为300μm。每20s给予一次刺激，刺激波宽是0.1ms。刺激强度为最大反应幅度的30％～40％时的刺激强度。当反应幅度稳定15分钟后灌流给药。
5、大鼠原代海马神经元电生理记录
体外培养10天左右的海马神经元，在外液中孵育30分钟后进行全细胞膜片钳记录。电极内液为葡糖酸钾内液。在I＝0模式记录膜电位。稳定记录10分钟。零镁外液为正常外液中去掉镁离子。PTX条件中，PTX浓度为100μM，KCl为6.5mM。
6、小鼠电刺激点燃模型的建立
C57小鼠麻醉后用立体定位仪固定头部。利用前囟定位左侧杏仁核(P:-0.12，L:+0.28，D:-0.46)，埋入电极。右侧颅骨钉入螺丝，其中一根缠绕地线。用牙科水泥固定后恢复7天，进行阈值测定。刺激频率为60Hz，波宽为1ms，刺激时长1s。每天以阈值强度刺激小鼠，并记录行为级别和后电位的长度，直至动物发至完全点燃(发生3次5级及定义为完全点燃)。癫痫行为学指标参照Racine分级法(1972)：0级，小鼠未见行为变化；1级，面部抽搐，胡须抖动；2级，不自主点头与规律性眨眼；3级，单侧前肢僵直并抬起；4级，双侧前肢抬起，直立；5级， 后肢抬起，直立并痉挛，失去姿势控制。
7、小鼠匹鲁卡品(pilocarpine)模型的建立
C57BL/6成年雄性小鼠腹腔先注射剂量为2mg/kg的东莨菪碱(Scopolamine methyl nitrate，Sigma)用以阻断匹鲁卡品的副作用，30分钟后注射剂量为300mg/kg的匹鲁卡品(Pilocarpine hydrochloride(Sigma)溶于0.9％(w/v)生理盐水中)，记录小鼠第一次发作至5级癫痫的时间，当小鼠维持持续性癫痫(status epilepticus)2小时后，腹腔注射剂量为4mg/kg的安定(Diazepam，Sigma)。
实施例
实施例1、SHH信号通路影响谷氨酸水平
为了研究SHH通路是否影响和胞外谷氨酸水平，本发明人利用高效液相色谱(HPLC)的方法检测了体外培养的海马神经元(分离大鼠海马原代培养)胞外的谷氨酸浓度，发现加入SHH(购自Sigma)可以促进谷氨酸堆积，而该作用能被此信号通路的抑制剂Cyclopamine(环巴胺，购自Sigma)所抑制(图1A，其中SHH浓度500ng/ml，Cyclopamine 10μM)。
随着SHH的浓度升高谷氨酸水平同时升高，在SHH达到500-1000ng/ml时谷氨酸水平达到峰值(图1B)，其升高水平与TBOA作用类似(图1C，其中SHH浓度500ng/ml，TBOA浓度100μM)。
为了进一步探索SHH通路如何影响和胞外谷氨酸水平，本发明人用3H标记谷氨酸的方法，观察SHH是否影响谷氨酸的摄取。实验结果显示，SHH与谷氨酸转运体的抑制剂TBOA作用类似，可以显著抑制细胞对于谷氨酸的摄取能力，且该作用同样是SHH浓度依赖的(图1D-F；D中SHH浓度500ng/ml，Cyclopamine浓度10μM；F中，DHK浓度300μM SHH浓度500ng/ml，TBOA浓度100μM)。
实施例2、Cyclopamine和GDC-0449可以抑制原代培养的海马神经元癫痫样放电
为了研究内源性SHH的作用，本发明人摸索了SHH释放的条件。ELISA的实验结果显示，用不含镁离子的细胞外液刺激体外培养的神经元15分钟，30分钟和60分钟都可以检测到SHH分泌量的显著升高(图2A)。
相类似的，本发明人发现在孵育同时给予GABA抑制剂(Picrotoxin)和高钾 离子含量的外液时，也能自15分钟起在外液中检测到SHH分泌量的上升(图2B)。
进一步，本发明人全细胞膜片钳记录的方法，在预孵育无Mg2+外液或是PTX，高浓度KCl外液30分钟后，观测神经元的放电情况，发现这两种刺激均可以体外诱导神经元自发癫痫样放电，而正常外液中几乎没有此类异常放电。并且此两种条件下，诱导产生的癫痫样放电的频率可以被Cyclopamine抑制(图3A，B)。相类似地，GDC-0449也可以抑制PTX诱导的癫痫样放电频率(图3C)。
实施例3、Cyclopamine可以降低大鼠急性脑片海马神经元群峰电位的幅度
为了探索在接近在体的情况下，Cyclopamine能否抑制癫痫发生，本发明人采用成年大鼠急性离体脑片电生理记录的方法。首先在孵育脑片的人工脑脊液中加入Picrotoxin(100μm)，并提高钾离子浓度(6.5mM)来诱导癫痫发作，ELISA结果显示有SHH大量的释放。在同样的情况下，本发明人在海马CA1胞体层胞外记录刺激诱导的多级群峰电位。在灌流Cyclopamine后，群峰电位的幅度均有下降。对第一个群峰电位的幅度进行统计，在灌流15分钟后有显著性差异(图4)。
上述结果提示，Cyclopamine有降低癫痫发作强度的作用。
实施例4、Cyclopamine在动物模型中抑制癫痫发作
为了进一步了解Cyclopamine是否在癫痫发生发展过程中起保护作用，本发明人参照文献Tan GH,et al.(2012)Neuregulin 1represses limbic epileptogenesis through ErbB4in parvalbumin-expressing interneurons.Nature neuroscience15(2):258-266，建立了小鼠电刺激点燃的癫痫模型。连续给与小鼠阈值刺激10天后，对照组小鼠可被完全点燃，而腹腔注射Cyclopamine的小鼠则需要20天才能达到完全点燃。且注射Cyclopamine小鼠在点燃过程中相比对照组小鼠都表现出更低的癫痫发作等级，和更短的后电位持续时间(图5)。
上述结果说明，Cyclopamine可以减轻或推迟癫痫的发作。
实施例5、GDC-0449在动物模型中抑制癫痫发作
Vismodegib(GDC-0449)(购自Selleck)是针对于Smo的，SHH通路另一种特异性抑制剂。由于其较少的副作用和良好的治疗效果，GDC-0449已于2012年1月30日获得美国FDA批准的用于治疗基底细胞瘤。截止至2011年6月，该药物还在进行与多种癌症，包括胃癌，胰腺癌，髓母细胞瘤，小细胞肺癌，软骨细胞 瘤，结肠癌，治疗的临床实验。
为了进一步验证SHH通路在癫痫情况的作用并为之后进行临床实验做准备，本发明人在体外培养的海马神经元以及小鼠电刺激点燃中，检测了GDC-0449的作用。结果发现GDC-0449与Cyclopamine有相类似的效果，同样可以延缓癫痫发生，抑制癫痫样放电。
实施例6、药物筛选
细胞水平筛选1
取海马神经元细胞，该细胞表达SHH信号通路的相关分子。将该种细胞作为用于筛选抑制SHH信号通路的药物的细胞模型。
测试组：用候选物质处理的上述细胞的培养物；
对照组：不用候选物质处理的上述细胞的培养物。
在处理后适当时间，采用常规方法测定所述细胞中SHH信号通路的激活情况。参照文献Jin Jiang,et al.(2008)Hedgehog Signaling in Development and Cancer,Developmental Cell 15:801-812(记载了在SHH信号通路中，下游分子的磷酸化水平，如Smo的磷酸化等，或是下游转录因子的表达量水平如Gli1、Gli2等，可以特异地提示该通路的激活情况)，如果与对照组相比，测试组中的Smo磷酸化水平或是Gli1/Gli2的mRNA或蛋白表达量显著下降20％以上，则说明该候选物质是潜在的治疗癫痫的物质。
采用Cyclopamine、GDC-0449作为候选物质进行了测试，结果发现，该处理可使Gli1、Gli2的mRNA及蛋白水平显著下降，提示SHH信号通路的活性显著下调，从而Cyclopamine、GDC-0449是潜在的治疗癫痫的物质。
细胞水平筛选2
取海马神经元细胞，该细胞表达SHH信号通路的相关分子。将该种细胞作为用于筛选抑制SHH信号通路的药物的细胞模型。
测试组：用候选物质处理的上述细胞的培养物；
对照组：不用候选物质处理的上述细胞的培养物。
在处理后适当时间，采用常规方法测定所述细胞的SHH蛋白的表达量、活性或分泌量。如果与对照组相比，测试组中的SHH蛋白的表达量、活性或分泌量显著下降，则说明该候选物质是潜在的治疗癫痫的物质。
实施例7、环巴胺在动物模型中抑制自发性癫痫的形成
为了进一步观察环巴胺(Cyclopamine，简称Cyclo)抑制癫痫发生发展的作用是否在不同模型间具有普适性，除了建立小鼠电刺激点燃的癫痫模型外，本发明人还又参照文献Tan GH，et al.(2012)Nature neuroscience 15(2):258-266，建立了小鼠匹鲁卡品(pilocarpine)模型，该模型为观察小鼠自发性癫痫的经典模型。
建模成功的小鼠被随机分成两组，其中一组作为空白对照每两天腹腔注射环巴胺的溶剂：45％HBC，另一组作为实验组每两天腹腔注射剂量为10mg/kg的环巴胺。录像结合人工记录建模后第4、6、8周小鼠自发性癫痫的发作等级和发作频率，每只小鼠每天观察7小时，共观察15天，癫痫行为学分级与小鼠电刺激点燃模型相同。实验结果显示，腹腔注射环巴胺的小鼠相对于对照组小鼠，自发性癫痫行为的发作频率在各个等级中都有下降趋势，其中严重的自发性癫痫行为(4-5级)发生频率下降更为显著。统计在观察时间内至少发作过一次5级癫痫行为的小鼠比例，对照组约为给药组的3倍，如图6。
上述结果说明，环巴胺可以显著地减轻或推迟自发性癫痫的发作。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (9)

  1. 一种SHH信号通路抑制剂的用途，用于制备改善或治疗癫痫的组合物。
  2. 如权利要求1所述的用途，其特征在于，所述改善或治疗癫痫包括：
    延缓癫痫发生；
    抑制海马神经元癫痫样放电；和/或
    降低癫痫发作强度。
  3. 如权利要求1所述的用途，其特征在于，所述的SHH信号通路抑制剂包括：环巴胺，GDC-0449，或它们的类似物。
  4. SHH信号通路的用途，其特征在于，用于筛选改善或治疗癫痫的潜在物质。
  5. 一种筛选改善或治疗癫痫的潜在物质的方法，所述方法包括：
    (1)用候选物质处理包含SHH信号通路的体系；
    (2)检测所述体系中SHH蛋白的表达量、活性或分泌量，或检测SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量；
    其中，若所述候选物质可降低SHH蛋白的表达量、活性或分泌量，或降低SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量，则表明该候选物质是改善或治疗癫痫的潜在物质。
  6. 如权利要求5所述的方法，其特征在于，步骤(1)包括：在测试组中，将候选物质加入到包含SHH信号通路的体系中；和/或
    步骤(2)包括：检测测试组的体系中SHH蛋白的表达量、活性或分泌量，或检测SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量，并与对照组比较，其中所述的对照组是不添加所述候选物质的包含SHH信号通路的体系；
    如果测试组中SHH蛋白的表达量、活性或分泌量在统计学上低于对照组，或SHH信号通路下游分子的磷酸化水平或SHH信号通路下游转录因子的表达量显著下降，就表明该候选物是改善或治疗癫痫的潜在物质。
  7. 如权利要求5或6所述的方法，其特征在于，所述的体系是细胞体系。
  8. 如权利要求7所述的方法，其特征在于，所述的体系是内源存在SHH信号通路的海马神经元或其细胞培养物。
  9. 一种用于改善或治疗癫痫的组合物，其特征在于，所述的组合物含有：
    (1)环巴胺，GDC-0449，或它们的类似物；和
    (2)药学上可接受的载体。
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