CN114984020A - Use of adenylate cyclase inhibitors for the treatment of epilepsy and related disorders - Google Patents
Use of adenylate cyclase inhibitors for the treatment of epilepsy and related disorders Download PDFInfo
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- CN114984020A CN114984020A CN202210463036.8A CN202210463036A CN114984020A CN 114984020 A CN114984020 A CN 114984020A CN 202210463036 A CN202210463036 A CN 202210463036A CN 114984020 A CN114984020 A CN 114984020A
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- 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
Abstract
The invention provides the use of an adenylate cyclase inhibitor for the preparation of a medicament for the treatment of epilepsy and related disorders. In particular to the application of a medicament NB001 for inhibiting AC1 and a medicament UBP310 for inhibiting a kainic acid receptor and a composition thereof in preparing a medicament for treating epilepsy. The invention uses pharmacology, electrophysiology, genetics, behaviourology and other methods to disclose the role of ACC to striatal cortical striatal circuit in epilepsy and the function of KA receptors in this circuit. Epileptiform activity leads to up-regulation of the action of KA receptors. The results indicate that KA receptors and AC1 are potential therapeutic targets for antiepileptics and neuroprotective agents, and AC1 inhibitors and KA can be used to treat epilepsy.
Description
Technical Field
The present invention relates to the use of an adenosine cyclase inhibitor and a Kainate receptor (KAR) antagonist in the preparation of a medicament for the treatment of epilepsy.
Background
Epilepsy is a destructive nervous system disease, which can lead to brain dysfunction and death in severe cases, and is commonly called as epilepsy or epilepsy. To date there are approximately 5000 million epileptic patients worldwide. The occurrence mechanism of the disease is very complex and has the characteristics of repeatability, paroxysmal property and the like: seizures can be caused by any form of brain injury, including structural and metabolic abnormalities of the brain tissue or changes in key molecules. Considering that the research on epileptogenesis and therapeutic approaches has been a research challenge in neuroscience, we believe that it is critical in basic and clinical research to enhance the study of the neurological mechanisms of epileptogenesis to deepen understanding of the disease and consequently to obtain effective therapies. Changes in neuronal excitability or excitability-inhibition balance have been shown to be the major mechanism of seizures, and the molecular determinants for such changes remain to be determined. Seizures may be induced acutely by blocking inhibitory synaptic transmission or activating excitatory synaptic transmission. The current antiepileptic drugs that can effectively control epileptic seizures act mainly on voltage-gated Na + Channel or Ca 2+ A channel to alter neuronal excitability. However, although these antiepileptic drug treatments reduce global neuronal excitability in the brain, the only available drug treatments target their symptoms only, and do not more naturally eliminate the occurrence of epilepsy (percucaand Meador, 2005; golden enberg, 2010). Therefore, there is a great need to find alternative anti-epileptic drug targets.
Temporal Lobe Epilepsy (TLE) is a common type of epilepsy in humans. Recurrent seizures originate in the medial or lateral temporal lobe, e.g., the hippocampus. Few studies have also reported that recurrent seizures may spread to a variety of structures, including the amygdala, the striatum, and the neocortex (Lothman et al, 1991). Frontal lobe epilepsy is the second common type of seizure, but is difficult to study due to its involvement in functions such as motivation, social interaction, and cognition. The Anterior Cingulate Cortex (ACC) is considered part of the limbic frontal cortex and is one of the most difficult brain regions to detect seizures. Therefore, clinical and scientific researchers still face a great challenge in order to effectively control the incidence of epilepsy.
The kainate receptor (KAR) consists of a family of ionotropic glutamate receptors, which consists of a combination of five subunits: GluK1-GluK 5. The glutamic acid analog kainic acid is a potent neurotoxin that has long been known to induce behavioral and electrophysiological seizures by activating kainic acid receptors. Kainic acid has been widely used in experiments to induce acute brain attacks and, as a chronic model of TLE, has similar characteristics after repeated treatments as observed in human patients with TLE. Kainic acid injection produces acute epileptogenesis mediated by karma and Marques, inhibition of presynaptic GABA release mediated by KAR and postsynaptic KAR activation of glutamatergic neurons (2013). Several studies have shown that the KAR receptor subunit GluK1 plays an important role in seizure activity (Falcon-Moya et al, 2018; Schubert and Albrecht, 2008; Smolders et al, 2002). In rat amygdala sections, high concentrations of ATPA induced spontaneous epileptiform outbreaks (Li et al, 2001), and GluK1 antagonists prevented hippocampal seizures induced by pilocarpine or electrical stimulation in rats (Smolders et al, 2002). Systemic administration of ATPA in vivo actually induced seizures in the hippocampus and amygdala. In GluK1 -/- This effect is abolished in mice (Fritsch et al, 2014). However, the role of KAR in epileptogenic activity remains a matter of debate. It has not been elucidated whether KAR is activated as an agonist to mediate seizure induction or whether the effect of KAR is directly upregulated under epileptogenic effects. There is currently no anticonvulsant therapy based on KAR signaling used in humans.
Adenylate Cyclase (AC), a class of membrane-integrated proteins, converts Adenosine Triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) and thereby elicits a cellular signaling response. In the body, various important physiological functions such as learning and memory, chronic pain, mood regulation andplays a regulating role in drug addiction. AC mainly comprises two major families: transmembrane ACs (AC1-9) and soluble AC (AC 10). The differences in unique organ and cell distribution characteristics and activation mechanisms allow each AC subtype to play a different physiological role in biological systems. Of these 10 subtypes, AC1 and AC8 are involved in the regulation of Ca 2+ The key AC subtype that reacts with CaM (Xia Z et al, 1997). And AC1 vs Ca, compared to AC8 2+ The increase in (c) is more sensitive. AC is widely distributed in mammalian cell membranes, and in ACC, AC1 is highly expressed in neurons of each layer (Wei F et al, 2002). In ACC, AC1 is selective for the modulated response. Deletion of AC1 had no effect on basal glutamate synaptic transmission, but blocked TBS or mating stimulus-induced LTP responses, whereas AC8 knockout mice did not block LTP production. Its selective inhibitor, NB001, was effective in blocking early LTP production in pyramidal cells. It was found that AC1 mediates behavioral sensitization to pain and enhancement of the spinal cord. It not only contributes to inflammation-induced activation of cAMP response element protein (CREB) but also promotes changes in long-acting excitatory synapses in spinal cord dorsal horn and transport of AMPA receptors. In these processes, AC1 is accomplished by activating a series of downstream molecular signaling pathways. Among these molecules, the Protein Kinase A (PKA) and the Protein Kinase C (PKC)/calmodulin-dependent protein kinase type II (CaMK II) signaling pathway play an important role. Likewise, AC1 is taken as Ca 2+ The activation of a signaling protein downstream of the KA receptor, whose role in epilepsy is unknown, and whether his inhibitors have a role in the modulation of epilepsy has yet to be investigated.
The present inventors have discovered that Pentylenetetrazol (PTZ) -induced seizure activity increases Kainic Acid Receptor (KAR) -mediated excitatory postsynaptic currents (EPSCs) in the cortico-striatal junctions from ACCs. These increases are dependent on the calcium-stimulated adenylate cyclase subtype 1 downstream of the KAR (AC 1). GluK 1-/-and AC 1-/-knockout mice attenuate seizures. In addition, intraperitoneal injection of NB001 dose-dependently inhibited seizure activity. Finally, the AC1 inhibitor analogs compound a and compound B also have an inhibitory effect on epilepsy. The present invention provides direct evidence for the treatment of epilepsy with KAR and adenylate cyclase inhibitors and reveals potential new anti-epileptic strategies.
Disclosure of Invention
The object of the present invention is to provide a new use of adenosine cyclase inhibitors or KAR antagonists in the treatment of epilepsy.
The purpose of the invention is realized as follows: use of a medicament that inhibits AC1 in the manufacture of a medicament for the treatment of epilepsy.
The medicament for inhibiting AC1 is NB001 or a pharmaceutically acceptable salt or solvate thereof; the NB001, 5- ((2- (6-amino-9-H-purin-9-yl) ethyl) amino) pentane-1-pentanol, has the structural formula:
the medicament for inhibiting AC1 comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically acceptable inert carrier material, organic or inorganic.
Suitable pharmaceutically acceptable organic or inorganic inert carrier materials include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
The pharmaceutical dosage form for inhibiting AC1 is in the form of tablet, coated tablet, suppository, capsule, or in the form of solution, suspension, or emulsion.
The medicament for inhibiting AC1 optionally comprises a preservative, stabilizer, wetting or emulsifying agent, salt for altering osmotic pressure, or a buffer.
The dosage of the active ingredients for the administration of the medicament for inhibiting AC1 is as follows: 1ng to 500 mg/kg.
The object of the invention is also achieved in that: use of an agent that inhibits a kainic acid receptor in the manufacture of a medicament for the treatment of epilepsy.
The agent of the kainic acid receptor is GluK1 receptor antagonist UBP310, wherein UBP310 is 1(2H) -pyrimidepropanoic acid, alpha-amino-3- [ (2-methoxy-3-thienyl) methyl ] -3,4-dihydro-5-methyl-2,4-dioxo-, (alpha S) and has the structural formula:
the agent for kainic acid receptors comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically acceptable inert carrier material, organic or inorganic.
Suitable pharmaceutically acceptable organic or inorganic inert carrier materials include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
The medicament dosage form of the kainic acid receptor exists in solid state in the form of tablets, coated tablets, suppositories and capsules, or exists in liquid state in the form of solution, suspension or emulsion.
The agent of the kainic acid receptor optionally contains preservatives, stabilizers, wetting or emulsifying agents, salts for varying the osmotic pressure, or adjuvants for buffers.
The dose of the active ingredients of the medicament for the kainic acid receptor is as follows: 1ng to 500 mg/kg.
The object of the invention can also be achieved by: a pharmaceutical composition for treating epilepsy comprises two compounds, NB001 and UBP 310.
The pharmaceutical composition for the treatment of epilepsy comprises an active ingredient for enteral or parenteral administration, and a suitable pharmaceutically acceptable inert carrier material, organic or inorganic.
Suitable pharmaceutically acceptable organic or inorganic inert carrier materials include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
The pharmaceutical composition for treating epilepsy is in the form of tablets, coated tablets, suppositories and capsules, and exists in a solid state, or exists in a liquid state in the form of solution, suspension or emulsion.
The pharmaceutical composition for treating epilepsy optionally comprises a preservative, a stabilizer, a wetting or emulsifying agent, a salt for changing osmotic pressure, or an adjuvant of a buffering agent.
The pharmaceutical composition for treating epilepsy comprises the following pharmaceutical active ingredients in percentage by weight: 1ng-500 mg/kg.
The invention has the following beneficial effects: the combination of the invention uses pharmacological, electrophysiological, genetic and behavioural methods to reveal the role of ACC in the cortical striatal loop of the striatum in epilepsy and the function of the KA receptor in this loop. Epileptiform activity leads to up-regulation of the action of KA receptors. The results indicate that KA receptor and AC1 are potential therapeutic targets for antiepileptics and neuroprotective agents, and AC1 inhibitors and KA can be used to treat epilepsy.
Drawings
Figure 1 increases the magnitude of KAR-mediated EPSCs in Pentylenetetrazol (PTZ) -induced epileptic mice, and gabapentin, a positive epilepsy treatment drug, can block the magnitude of KAR-mediated EPSCs.
Figure 2 PTZ-induced epilepsy was significantly reduced in GluK 1-/-and AC 1-/-mice, but not in AC 8-/-mice.
Figure 3 dose-dependent attenuation of PTZ-induced epilepsy by the inhibitor NB001 with AC1 is superior to gabapentin.
Figure 4 therapeutic effect of the AC1 inhibitor analogs compound a and compound B on PTZ-induced epilepsy.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings. However, these examples are only for explaining the technical solution of the present invention, and they are not to be construed as limiting the scope of the present invention.
It should be noted that the technical features of the present invention are described in the specification only in conjunction with the respective specific embodiments, however, the technical features are not limited to the specific embodiments. The present invention also includes any combination of all of the above-described features in various embodiments, unless otherwise specified.
Unless otherwise indicated, the following examples are carried out according to conventional methods known to those skilled in the art using reagents of the grade commonly used in the corresponding assays and preparations in the art.
In one embodiment of the invention, the invention relates to the use of an antagonist that inhibits the kainic acid receptor in the manufacture of a medicament for the treatment of epilepsy.
In one embodiment of the present invention, wherein said agent inhibiting the kainic acid receptor is GluK1 receptor antagonist UBP 310.
In one embodiment of the invention, the invention relates to the use of a medicament that inhibits AC1 in the manufacture of a medicament for the treatment of epilepsy.
In one embodiment of the present invention, wherein the agent that inhibits AC1 is NB001 or a pharmaceutically acceptable salt, solvate thereof.
In one embodiment of the present invention, wherein said epilepsy and related diseases or conditions are selected from the group consisting of: status epilepticus, generalized periodic epileptiform discharges, periodic lateral epileptiform discharges, and seizures.
In one embodiment of the invention, the subject is administered orally or parenterally. The compounds described herein may be formulated for intranasal, buccal, intravenous, intramuscular, subcutaneous, intramural, mucosal, sublingual, intraocular and topical administration.
In one embodiment of the invention, the medicament of the invention is administered within 10 hours, 8 hours, 5 hours, 3 hours, 1 hour or 0.5 hours after a seizure, such as a status epilepticus seizure, such as a refractory status epilepticus seizure, has begun. In some embodiments, the medicament of the invention is administered within 60 minutes, 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes after a seizure, such as a status epilepticus seizure, such as a refractory status epilepticus seizure, has begun. In some embodiments, the medicament of the invention is administered after a seizure, such as a status epilepticus seizure, such as a refractory status epilepticus seizure, has lasted for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes.
In another embodiment, the medicament of the invention is administered prior to the initiation of a seizure, such as a status epilepticus seizure, such as a refractory status epilepticus seizure.
For the use of the compounds of the invention as medicaments, the medicaments should take the form of pharmaceutical preparations which, in addition to the active ingredients for enteral or parenteral administration, comprise suitable pharmaceutically, organic or inorganic inert carrier materials, such as water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols and the like. The pharmaceutical preparations can be present in solid form, for example as tablets, coated tablets, suppositories, capsules, or in liquid form, for example as solutions, suspensions or emulsions. In addition, they optionally contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, salts for varying the osmotic pressure, or buffers.
In one embodiment, the compounds of the present invention may be administered at a dose of: 1ng to 500 mg/kg.
Additionally, a therapeutically effective dose of a compound of the invention may be: 1ng-500 mg/kg.
The pharmaceutical composition of the present invention may comprise the following agents in amounts: 1ng-500 mg.
Example (b):
the present invention will be described in further detail with reference to the following examples.
Experimental animals: adult C57BL/6 mice (8 to 12 weeks old). GluK 1-/-mice, AC 1-/-mice and AC 8-/-mice were laboratory self-bred breeding mice with C57BL/6 as background. All mice maintained a 12 hour light/dark cycle with unlimited food and water supply.
Preparing brain slices: coronal brain sections (300 μm) containing ACC and dorsal striatum were prepared using the previous method (Sellmeijer et al, 2018; Song et al, 2017). Briefly, mice were anesthetized with isoflurane and sacrificed by decapitation. The entire brain was quickly removed from the skull and submerged in ice cold oxygen (95% O) 2 And 5% CO 2 ) In artificial cerebrospinal fluid (ACSF), comprising (in mM) 124 NaCl, 2.5 KCl, 2 CaCl 2 、2 MgSO 4 、25 NaHCO 3 、1 NaH 2 PO 4 And 10 glucose). For preparation of coronal brain sections, the brain was glued to a microtome (Leica, VT1200S) at the cutting stage. The slices were transferred to a bed with oxygen (95% O) at room temperature 2 And 5% CO 2 ) The immersion incubation of ACSF is continued untilOne hour less.
In vitro whole cell patch clamp recording: experiments were performed in a recording room with an Olympus BX51W1 microscope with infrared DIC optics for visualization of whole cell patch clamp recordings. Excitatory postsynaptic currents (EPSCs) were recorded from neurons in the striatum with Axon 200B amplifiers (Axon Instruments) and stimulation was delivered by bipolar tungsten stimulation electrodes placed in layers V/VI of the ACC. Recording pipettes (3 to 5M Ω) were filled with a solution (adjusted to pH 7.2 with KOH, 290mOsmol) containing (in mM) 145 potassium gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP and 0.1 Na 3-GTP. Throughout the experiment, EPSCs were recorded in a voltage clamp configuration and membrane potentials were held at-60 mV. In the presence of AP5 (50. mu.M), AMPA/KA receptor-mediated EPSC was induced by repeated stimulation at 0.02 Hz. Both AP5 and GYKI 53655(100 μ M) are applied for KA current. For frequency facilitation, repeated stimuli were delivered at 200Hz (5, 10 or 20 strokes). Kuduning (100 μ M) was consistently present to block GABA in all experiments A Receptor-mediated inhibitory synaptic current. The access resistance was 15 to 30 M.OMEGA.and was monitored throughout the experiment. If a resistance change of 15% is switched in during the experiment, the data is discarded. The data was filtered at 1kHz and digitized at 10kHz using digitata 1440A.
Induction of in vivo seizure activity: as previously reported, pentylenetetrazol (PTZ; Sigma, MO, USA) at 50mg/kg was administered intraperitoneally to induce seizures in vivo. Prior to PTZ injection, mice were removed from their home cages and individually placed into test cages of new plastic cages with corn cob liners for 30 minutes. Immediately after injection, mice were returned to test cages for video recording of seizure activity. Seizure activity, typically induced within 15 minutes of PTZ injection, was observed within 30 minutes after injection. Seizure behavior was scored based on the Racine scale (Ferraro et al, 1999; Naydenov et al, 2014): stage 0, no seizures; stage 1, hypomotility, nodulation; stage 2, Partial Clonus (PC) includes clonic episodes affecting the face, head or forelimbs; stage 3, Generalized Clonus (GC), including whole body clonus of all limbs and tails, standing up with the hind legs or falling down; stage 4, generalized tonic clonic seizures (GTC), jumps, screaming, falls; stage 5, severe tics, maximal seizures of tonic clonic are associated with spontaneous recovery of the mouse; stage 6, tonic seizures followed by death.
Seizure susceptibility is calculated from the latency of Partial Clonic (PC), Generalized Clonic (GC) and Generalized Tonic Clonic (GTC) seizures, as previously described (Ferraro et al, 1999; Naydenov et al, 2014).
Susceptibility to seizure was 0.2 ═ 0.3 ═ 0.5 ═ 0.3 ═ 1/GC latency) + (1/GTC latency).
Chemical and pharmaceutical applications: d (-) -2-amino-5-phosphonopentanoic acid (AP5), 1- (4-aminophenyl) -3-methylcarbamoyl-4-methyl-3, 4-dihydro-7, 8-methylenedioxy-5H-2, 3-benzodiazepine
Hydrochloride (GYKI 53665) and UBP310 were obtained from Tocris Cookson (Bristol, UK). Kukonin (PTX) and Pentylenetetrazol (PTZ) were obtained from Sigma Ardrich (Canada). NB001 was supplied by NeoBrain Pharmac Inc (Canada). The drug was prepared as a stock solution for frozen aliquots at-20 ℃. All these drugs were diluted from stock solutions to the final desired concentration in ACSF before application to the perfusion solution.
And (3) data analysis: data were collected and analyzed using the clautex 10.2 and claupifit 10.2 software (Molecular Devices). For comparison between the two groups, either unpaired student's t test or paired t test was used. For comparisons between the three groups, one-way ANOVA or two-way ANOVA was used (Student-Newmann-Keuls or Tukey test was used for post-hoc comparisons). All data are expressed as mean ± Standard Error of Mean (SEM). In all cases, P <0.05 was considered statistically significant.
Results 1: following an increase in vitro seizure-like activity in brain slices in the field of in vivo Pentylenetrazole (PTZ) -induced behavioral seizures, it was tested whether the KAR-mediated EPSCs were also altered in an in vivo seizure model. As previously reported (Krishnan et al, 2017; Naydenov et al, 2014), GABA was used A Receptor antagonist pentylenetetrazole(PTZ) induced in vivo seizure model. In wild type mice, intraperitoneal (i.p.) injection of PTZ (50mg/kg) can successfully induce behavioral seizures in vivo 2 to 5 minutes after injection. KAR-mediated EPSCs were recorded in brain sections 0.5 hours after onset of PTZ induction. In the presence of bittonin and AP5, there was small residual EPSCs after GYKI 5365510 minutes of administration in ACC-striatal synaptic connections after PTZ-induced onset. KAR-mediated EPSCs were significantly increased and KA receptors contributed 16.1 ± 1.3% of AMPA/KAR-mediated EPSCs in the PTZ group compared to 6.6 ± 0.4% in the saline group (fig. 1B and 1C). Furthermore, consistent with the results of in vitro seizure-like activity in brain sections, repeated stimulation also significantly increased KAR-mediated EPSC (F (1,60) ═ 28.66, P, compared to saline controls in the PTZ-induced seizure model<0.001, two-way ANOVA, fig. 1C). In the PTZ-induced seizure group, the KAR-mediated input-output curve of EPSC was significantly shifted to the left (F (1,70) ═ 11.37, P<0.01, two-way ANOVA, fig. 1D). However, there was no significant change in KAR-mediated EPSC when the antiepileptic, gabapentin (50mg/kg, i.p.), was administered 30 minutes before PTZ (16.1 ± 1.3%, n ═ 7 neurons/3 mice). There was no difference in KAR-mediated input-output curves of EPSCs in the PTZ group compared to the control group (P)>0.05, fig. 1D), although repeated stimulation appears to increase KAR-mediated EPSC (P)>0.05, fig. 1C). Taken together, these results indicate that both in vitro and in vivo priming-like activity up-regulates KAR-mediated EPSCs in the ACC-striatal pathway.
Results 2: knockout of AC1 function attenuated seizure activity in vivo: the above results indicate that KA receptors and AC1 may contribute to seizure activity. To verify whether inhibition of GluK1 and AC1 function could reduce behavioral seizures in vivo, PTZ-induced seizures were studied in GluK 1-/-mice, AC 1-/-mice and AC 8-/-mice (fig. 2). PTZ (50mg/kg, i.p.) induces seizures that develop from the hypoactive (stage 1), local (PC, stage 2), extensive (GC, stage 3) and generalized tonic (GTC, stage 4) stages of the seizure. The largest tonic clonic seizures occasionally led to death in a few mice (stage 6), or were associated with spontaneously recovering mice (stage 5). As shown in figure 2A, PTZ-induced seizure severity scores were reduced in both GluK 1-/-and AC 1-/-mice, but not in AC 8-/-mice (WT: 4.87 ± 0.24, n ═ 15 mice; GluK 1-/-4.20 ± 0.23, n ═ 15 mice, P < 0.05; AC 1-/-3.50 ± 0.44, n ═ 16 mice, P < 0.05; AC 8-/-4.60 ± 0.25, n ═ 11 mice, P > 0.05; fig. 2a). Furthermore, the susceptibility to attacks was also significantly reduced in GluK 1-/-and AC 1-/-mice, but not in AC 8-/-mice (WT: 0.52 + -0.08; GluK 1-/-0.33 + -0.03, P < 0.05; AC 1-/-0.14 + -0.03, P < 0.001; AC 8-/-0.32 + -0.02, P > 0.05; FIG. 2B). Next, the latency into different stages of onset in GluK 1-/-mice, AC 1-/-mice and AC 8-/-mice was analyzed. The time to enter the PC phase was prolonged in GluK 1-/-and AC 1-/-mice compared to wild type mice, but was unchanged in AC 8-/-mice (WT: 1.47 + -0.21 sec; GluK 1-/-: 2.58 + -0.15 sec, P < 0.001; AC 1-/-: 3.37 + -0.62 sec, P < 0.05; AC 8-/-: 1.80 + -0.25 sec, P > 0.05; FIG. 2C). In these three mice, the latency into GC and GTC phases did not change significantly. The reason may be that some mice failed to reach the GC or GTC stage in GluK 1-/-and AC 1-/-mice. Mortality in GluK 1-/-mice, AC 1-/-mice and AC 8-/-mice decreased slightly after PTZ injection, but still a small number of mice died (WT: 26.67%; GluK 1-/-13.33%; AC 1-/-18.75%; and AC 8-/-9.1%; FIG. 2D).
Results 3: inhibition of AC1 function attenuates seizure activity in vivo: in addition, the effect of gabapentin and the AC1 inhibitor NB001 on PTZ-induced seizure behavior was tested in wild-type mice. Pretreatment was performed 30 minutes prior to PTZ injection using saline, gabapentin (50mg/kg, i.p.) or NB001(2, 10, 20 and 50mg/kg, i.p.). Gabapentin was found to have a significant anti-epileptic effect. Gabapentin reduced seizure severity scores (saline: 4.92 ± 0.30, n-12; gabapentin: 4.08 ± 0.33, n-12; P <0.05) and PTZ-induced seizure susceptibility (saline: 0.45 ± 0.02; gabapentin: 0.31 ± 0.03, P <0.05, fig. 3A and 3B). Gabapentin also reduced overall seizure-related mortality (1 death in a sample of 12 mice, fig. 3D). However, the use of gabapentin did not significantly prolong the latency into PC, GC and GTC phases induced by PTZ (fig. 3C). After pretreatment with NB001, an AC1 inhibitor, NB001 was found to dose-dependently reduce the severity of seizure behavior (2 mg/kg: 4.67 ± 0.47, n ═ 12; 10 mg/kg: 4.33 ± 0.47, n ═ 12; 20 mg/kg: 4.18 ± 0.30, n ═ 16, P < 0.05; 50 mg/kg: 3.44 ± 0.25, n ═ 16, P < 0.001; fig. 3A) and reduce seizure susceptibility in PTZ-induced seizure mice (2 mg/kg: 0.24 ± 0.03, P < 0.001; 10 mg/kg: 0.19 ± 0.02, P < 0.001; 20 mg/kg: 0.26 ± 0.04, P < 0.05; 50 mg/kg: 0.22 ± 0.02, P < 0.001; fig. 3B). Furthermore, in PTZ-induced seizure mice, NB001 significantly prolonged the latency into both PC and GC phases and reduced seizure-related mortality (fig. 3C and 3D). Taken together, these results indicate that both GluK1 and AC1 may serve as potential therapeutic targets for epilepsy and that the AC1 inhibitor NB001 has a potent therapeutic effect on seizure behavior.
Results 4: novel inhibitors of adenosine cyclase attenuate seizure activity in vivo. I.p. injection of compound a (30mg/kg) in mice (n-4 mice) was found to significantly reduce seizure-like activity by intraperitoneal injection of the novel inhibitor of adenylyl cyclase, compounds a and B, in the same manner as NB001 for its antiepileptic effect. Similar inhibition was found with the same dose (30mg/kg) of compound B. Thus, compounds a and B have been identified as novel inhibitors that inhibit the activity of both AC1 and AC 8.
Abbreviation table:
AC 1: adenylate cyclase subtype 1;
AC 8: adenylate cyclase subtype 8;
ACC: the front buckle is provided with a cortex;
ACSF: artificial cerebrospinal fluid;
AMPA: alpha-amino-3-hydroxy-5-methyl-4-iso
Oxazole-propionic acid;
NMDA: N-methyl-D-aspartic acid;
EPSC: excitatory postsynaptic current;
KAR: a kainic acid receptor;
PTZ: pentylenetetrazole.
Claims (22)
1. Use of a medicament that inhibits AC1 in the manufacture of a medicament for the treatment of epilepsy.
2. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 1, wherein the medicament inhibiting AC1 is NB001 or a pharmaceutically acceptable salt, solvate thereof; the NB001, 5- ((2- (6-amino-9-H-purin-9-yl) ethyl) amino) pentane-1-pentanol, has the structural formula:
3. use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 1 or 2, wherein the medicament inhibiting AC1 comprises an active ingredient for enteral or parenteral administration, and a suitable pharmaceutically acceptable inert carrier material, organic or inorganic.
4. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 3, wherein the suitable pharmaceutically acceptable inert organic or inorganic carrier materials include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
5. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 1 or 2, wherein the dosage form of the medicament inhibiting AC1 is in the form of a tablet, coated tablet, suppository, capsule in a solid state, or in the form of a solution, suspension or emulsion in a liquid state.
6. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 1 or 2, wherein the medicament inhibiting AC1 optionally comprises a preservative, stabilizer, wetting or emulsifying agent, salt to modify osmotic pressure, or adjuvant to a buffer.
7. Use of a medicament inhibiting AC1 according to claim 1 or 2 in the manufacture of a medicament for the treatment of epilepsy, wherein the medicament inhibits AC1 is administered at an active ingredient dose of: 1ng to 500 mg/kg.
8. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 5, wherein the medicament inhibiting AC1 optionally comprises a preservative, stabilizer, wetting or emulsifying agent, salt to modify osmotic pressure, or buffer.
9. Use of a medicament inhibiting AC1 in the manufacture of a medicament for the treatment of epilepsy according to claim 5, wherein the medicament inhibits AC1 is administered at an active ingredient dose of: 1ng to 500 mg/kg.
10. Use of an agent that inhibits a kainic acid receptor in the manufacture of a medicament for the treatment of epilepsy.
11. The use of an agent that inhibits the kainic acid receptor for the manufacture of a medicament for the treatment of epilepsy according to claim 10, wherein said agent is the GluK1 receptor antagonist UBP310, said UBP310 being 1(2H) -pyrimideppanoic acid, α -amino-3- [ (2-carboxy-3-thienyl) methyl ] -3,4-dihydro-5-methyl-2,4-dioxo-, (α S) having the structural formula:
12. use of a medicament inhibiting the kainic acid receptor according to claim 10 or 11 in the manufacture of a medicament for the treatment of epilepsy, wherein the medicament comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically acceptable organic or inorganic inert carrier material.
13. Use of an agent that inhibits kainic acid receptors for the manufacture of a medicament for the treatment of epilepsy according to claim 12, wherein said suitable pharmaceutically acceptable organic or inorganic inert carrier materials include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
14. Use of the agent for inhibiting the kainic acid receptor according to claim 10 or 11, wherein the agent for inhibiting the kainic acid receptor is in a solid state in a form of a tablet, a coated tablet, a suppository, a capsule, or in a liquid state in a form of a solution, a suspension, or an emulsion, for the preparation of a medicament for treating epilepsy.
15. Use of the agent inhibiting the kainic acid receptor for the manufacture of a medicament for the treatment of epilepsy according to claim 10 or 11, wherein the agent optionally comprises a preservative, stabilizer, wetting or emulsifying agent, salt to change osmotic pressure, or a buffer.
16. Use of an agent inhibiting the kainic acid receptor according to claim 10 or 11 for the manufacture of a medicament for the treatment of epilepsy, wherein the agent inhibiting the kainic acid receptor is administered at an active ingredient dose of: 1ng to 500 mg/kg.
17. A pharmaceutical composition for the treatment of epilepsy comprising two compounds NB001 and UBP 310.
18. A pharmaceutical composition for the treatment of epilepsy according to claim 17, comprising an active ingredient for enteral or parenteral administration, and a suitable pharmaceutically acceptable inert carrier material, organic or inorganic.
19. The pharmaceutical composition for the treatment of epilepsy according to claim 18, wherein said suitable pharmaceutically acceptable inert organic or inorganic carrier materials comprise water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
20. The pharmaceutical composition for treating epilepsy according to claim 17 or 18, wherein the pharmaceutical composition for treating epilepsy is in the form of tablet, coated tablet, suppository, capsule, and is in solid state, or in the form of solution, suspension or emulsion and is in liquid state.
21. The pharmaceutical composition for the treatment of epilepsy according to claim 17 or 18, wherein the pharmaceutical composition for the treatment of epilepsy optionally comprises a preservative, a stabilizer, a wetting or emulsifying agent, a salt for varying the osmotic pressure, or an adjuvant of a buffer.
22. The pharmaceutical composition for treating epilepsy according to claim 17 or 18, wherein the pharmaceutical composition for treating epilepsy comprises the following pharmaceutical active ingredients in percentage by weight: 1ng-500 mg.
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| Title |
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| DENISE K. GROSENBAUGH 等: "The Role of Kainate Receptors in the Pathophysiology of HypoxiaInduced Seizures in the Neonatal Mouse", SCIENTIFIC REPORTS, vol. 1, pages 10 - 15 * |
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