CN105853405B - Benzbromarone is preparing the application in voltage gated k+ channel blocker KCNQ agonist - Google Patents

Abstract

The present invention provides a kind of Benzbromarones to prepare the application in KCNQ agonist；In addition the present invention also provides a kind of Benzbromarones to prepare as the application in KCNQ anti-depressant medications；Wherein, in above-mentioned application, the KCNQ agonist can be used as KCNQ2 agonist, KCNQ2/Q3 agonist, KCNQ4 agonist and KCNQ5 agonist.Present invention show Benzbromarones to have enhancing KCNQ2, KCNQ2/Q3, KCNQ4 and KCNQ5 electric current；Enhance endogenous KCNQ electric current, adjust hippocampal neuron resting membrane electric potential level and action potential frequency, adjusts dorsal root ganglion neurons resting membrane electric potential level and action potential frequency.Animal experiments show that Benzbromarone significantly alleviates the mouse epileptic attack of maximal electroshock induction, it is obviously reduced the inflammatory pain of formalin, bradykinin and monosodium urate Salt treatment.

Description

Benzbromarone is preparing the application in voltage gated k+ channel blocker KCNQ agonist

Technical field

The invention belongs to drug fields, in particular it relates to Benzbromarone is preparing the application in KCNQ agonist, Wherein, in above-mentioned application, the KCNQ agonist can for KCNQ2 agonist, KCNQ2/Q3 agonist, KCNQ4 agonist and KCNQ5 agonist.In addition, the present invention also provides a kind of Benzbromarone in preparation as KCNQ agonist and endogenous KCNQ electricity Flow the application in anti-depressant medications, wherein in above-mentioned application, the KCNQ agonist can be KCNQ2 agonist, KCNQ2/Q3 Agonist, KCNQ4 agonist, KCNQ5 agonist or endogenous KCNQ electric current agonist.Moreover, it relates to benzene bromine horse The grand application in the disease treatment and prevention using the channel KCNQ as target spot, the disease include as epilepsy, anxiety, hypertension, Inflammatory pain, neuropathic pain etc..

Background technique

Benzbromarone (Benzbromarone, as shown in the following Expression 1), also known as benzbromarone, Benzbromarone, narcaricin, Benzbromarone, Allopurinol, Benzbromarone；It is benzofuran derivatives, chemical full name: (3,5- bis- bromo- 4- hydroxy phenyl) (2- ethyl -3- benzofuranyl) ketone.Benzbromarone is a kind of uricosuric object, is clinically widely used as gout treatment.

Gout be uric acid generate it is excessive and (or) underexcretion, lithate accumulates precipitating in joint and cartilaginous tissue A kind of caused arthritis disease (Roddy, E. etc., Arthritis Res Ther.2010,12 (6), 223).Benzbromarone is made It is main to treat gout by inhibiting renal tubule to reduce Plasma Uric Acid content to the reabsorption of uric acid for a kind of antigout prescription medicine (Rider TG etc. Rheumatology (Oxford) .2010,49 (1), 5-14).Previous research show that Benzbromarone enhancing is exhaled Inhale a kind of calcium ion activated chloride channel function of road epithelium, and imply its be possibly used for the treatment of asthma (Huang F etc., Proc Natl Acad Sci USA.2012,109 (40), 16354-9).But potassium up to the present, is acted on for Benzbromarone The physiological role of effect and performance on ion channel does not have relevant report.

Voltage gated k+ channel blocker (channel Kv) is to have now been found that most ion channel families, shares 12 members (K_V1.X-K_V12.X).The channel KCNQ is the 7th member (Kv7) in the channel Kv, including five hypotype KCNQ1~KCNQ5.KCNQ1 It is distributed mainly in cardiac system, with the I in complementary subunit KCNE1 composition myocardial action potential_KsIt is dynamic to be responsible for cardiac muscle for electric current Make potential repolarisation.KCNQ2~KCNQ5 is distributed mainly in maincenter and peripheral neverous system, also referred to as the nerve channel KCNQ, is adjusted Save level of membrane potential and nerve excitability (Brown, D.A. etc., Br J Pharmacol 2009,156 (8), 1185-95.)； The channel KCNQ4 and KCNQ5 is also distributed in the smooth muscle, skeletal muscle of Non nervous system, is adjusted by influencing level of membrane potential The contraction and diastole (Stott, J.B. etc., 2014,19 (4) 413-4. of Drug Discov Today) of muscle.

The neural channel KCNQ can be used as the therapeutic agent target spot of the psychoneurals excitability abnormal diseases such as epilepsy, mainly include Following evidence and advantage: first, KCNQ channel current activation threshold lower (subthreshold electronic potential about -60mV can be opened), activation are slow Slowly, also non-inactivation after activating, thus " brake " can be played in neuron epileptic paradoxical discharge and acted on, while maintaining neuron Normal reaction；Second, height expression in the channel KCNQ2 and KCNQ3 at aixs cylinder Start Fragment, so that adjusting KCNQ channel function to movement Electric spike plays a significant role；The channel the KCNQ2 and KCNQ3 mutation of third, maincenter distribution can lead to benign familial new life Youngster faints from fear (a kind of neonatal seizure disease)；4th, neural KCNQ channel agonist retigabine (Retigabine) in The adjuvant treatment of clinical part epileptic attack is used within 2011 by U.S. Food and Drug Administration (FDA) approval.More than Evidence shows that the neural channel KCNQ is the important target spot of the psychoneurals excitability abnormal diseases such as treatment and/or prevention epilepsy, KCNQ channel agonist be can be used for treating this kind of disease (Grunnet, M. etc., Eur J Pharmacol2014,726,133- 7.)。

The neural channel KCNQ can be used as therapeutic agent for pain target, mainly include following evidence: firstly, the nerve channel KCNQ is whole A sensation of pain, which conducts and experiences access, expression and distribution, influences the conduction felt.Secondly, heredity or pharmacology inhibit mind It will lead to neuronal excitability allergy through KCNQ electric current, and action potential granting can then be inhibited by enhancing nerve KCNQ electric current.Third, Animal is caused to feel that sensibility reduces to noxious stimulation after the neural channel KCNQ mutation or gene knockout；In addition, aching in animal In pain model, gives KCNQ channel agonist and inhibitor and mitigate and induce (or increase) ANIMAL PAIN behavior reaction respectively.With Above it turns out that, the refreshing channel KCNQ can be used as the target spot of pain therapy, and neural KCNQ channel agonist can be used for pain therapy Drug (Rivera-Arconada I etc., Front Mol Neurosci 2009,2,10).

It removes as neuropsychiatric disease therapy target and KCNQ channel agonist as related neural spirit disease in the channel KCNQ Outside the therapeutic agent of disease, the channel KCNQ is alternatively arranged as smooth muscle conditions, such as the therapy target of hypertension, asthma and antenatal throe (Stott, J.B. etc., 2014,19 (4) 413-4. of Drug Discov Today).Be distributed in KCNQ4 in smooth muscle cell and KCNQ5 pass effect level of membrane potential adjusts the opening of valtage-gated calcium channel on smooth muscle cell membrane, regulates and controls cellular calcium Ion concentration controls the contraction and diastole of smooth muscle.Give KCNQ channel agonist and inhibitor cause smooth muscle diastole and Shrink, KCNQ channel agonist drug Flupirtine (Flupirtine) have apparent hypotensive activity (Szelenyi, I., Inflamm Res 2013,62 (3), 251-8.).

The present invention utilizes whole cell protein electrophoresis, current clamp technique, mouse maximal electroshock epilepsy model, rats and mice good fortune Your Malin (Formalin), bradykinin and monosodium urate Salt treatment pain model, discovery Benzbromarone can enhance KCNQ2, The channel KCNQ2/Q3, the channel KCNQ4 and KCNQ5 channel function；Benzbromarone enhances nerve cell endogenous KCNQ channel function, Hippocampal neuron resting membrane electric potential level and action potential frequency are adjusted, it is horizontal to adjust dorsal root ganglion neurons resting membrane electric potential With action potential frequency；Benzbromarone significantly alleviates the mouse epileptic attack of maximal electroshock stimulation induction, alleviates by formal The rats and mice Pain behaviour of woods, bradykinin and monosodium urate Salt treatment reacts.

Summary of the invention

It is an object of the present invention to provide a kind of Benzbromarones to prepare the application in KCNQ agonist；

Wherein it is preferred to which the KCNQ agonist can be KCNQ2 agonist, KCNQ2/Q3 excitement in above-mentioned application Agent, KCNQ4 agonist and KCNQ5 agonist.

Further, it is another object of the present invention to provide a kind of Benzbromarone is in preparing the drug as KCNQ agonist Application；

Wherein it is preferred to which the KCNQ agonist can be KCNQ2 agonist, KCNQ2/Q3 excitement in above-mentioned application Agent, KCNQ4 agonist and KCNQ5 agonist；And, it is preferable that the drug can be used to treat or prevent the nerve essence such as epilepsy Refreshing disease treats or prevents the smooth muscle conditions such as hypertension, asthma, antenatal throe, treats or prevents non-syndrome and often dyes Body dominant heredity deafness disease, and/or treat or prevent inflammatory pain or locality or systemic neuropathic pain.

Further, it is another object of the present invention to provide a kind of Benzbromarone is in preparing endogenous KCNQ electric current agonist Application.

In addition, it is also another object of the present invention to provide a kind of Benzbromarone in preparation as the excitement of endogenous KCNQ electric current Application in the drug of agent；

Wherein, in above-mentioned application, the drug can be used to treat or prevent the neuropsychiatric diseases such as epilepsy, treatment or pre- It is deaf to treat or prevent non-syndrome autosomal dominant for the smooth muscle conditions such as anti-hypertension, asthma, antenatal throe Disease, and/or treat or prevent inflammatory pain or locality or systemic neuropathic pain.

Present invention show the Benzbromarone enhancing channel KCNQ2, the channel KCNQ2/Q3, the channel KCNQ4 and the channel KCNQ5 function Energy；Enhance endogenous KCNQ channel current, adjusts neuronal excitability；Zoopery shows that Benzbromarone alleviates epileptic attack And Pain behaviour.The invention shows Benzbromarones can be used as KCNQ channel agonist and endogenous KCNQ channel agonist, is used for Preparation treatment is using the channel KCNQ as the neuropsychiatric disease of target spot, the prevention of smooth muscle conditions and deaf disease or therapeutic agent.

Detailed description of the invention

Fig. 1 shows the figure that Benzbromarone influences KCNQ2 electric current in the embodiment of the present invention 1, in which:

Figure 1A is that current graph before and after 10 μM of Benzbromarones is administered in the channel KCNQ2；

Figure 1B is the dose-effect curve of the Benzbromarone in the channel KCNQ2；

Fig. 1 C is that conductance voltage curve before and after 10 μM of Benzbromarones is administered in the channel KCNQ2；

Fig. 1 D is that closing phase current homogenization in the channel KCNQ2 is bent before and after 10 μM of Benzbromarones of administration under+50mV test voltage Line chart；

Fig. 1 E is that phase time constant statistics is closed in the channel KCNQ2 before and after 10 μM of Benzbromarones of administration under+50mV test voltage Figure.

Fig. 2 shows the figure that Benzbromarone in the embodiment of the present invention 1 influences the different aggressiveness channel current of KCNQ2/Q3, In:

Fig. 2A is that current graph before and after 10 μM of Benzbromarones is administered in the different aggressiveness channel KCNQ2/Q3；

Fig. 2 B is that KCNQ2/Q3 closes phase current in different aggressiveness channel before and after 10 μM of Benzbromarones of administration under+50mV test voltage Uniform curve graph；

Fig. 2 C is that conductance voltage curve before and after 10 μM of Benzbromarones is administered in the different aggressiveness channel KCNQ2/Q3.

Fig. 3 shows in the embodiment of the present invention 2 Benzbromarone to dorsal root ganglion endogenous KCNQ electric current and action potential The figure of influence, in which:

Fig. 3 A is before 10 μM of Benzbromarones and endogenous KCNQ current inhibitor XE991 is administered in endogenous KCNQ sequence of currents Current graph afterwards；

Fig. 3 B is that the lower 10 μM of Benzbromarones of -20mV test voltage enhance endogenous KCNQ electric current statistical chart；

Fig. 3 C is that Dorsal Root Ganglion Neurons inject 400pA electric current 10 μM of Benzbromarones of administration and endogenous KCNQ electricity Flow inhibitor XE991 influences action potential and provides frequency diagram；

Fig. 3 D is that Dorsal Root Ganglion Neurons inject 400pA electric current 10 μM of Benzbromarones of administration and endogenous KCNQ electricity Flow inhibitor XE991 action potential provides number statistical figure.

Fig. 4 shows in the embodiment of the present invention 3 that Benzbromarone is horizontal to hippocampus of rats resting membrane electric potential and movement The figure that current potential influences, in which:

Fig. 4 A is that hippocampus of rats injects 400pA electric current 10 μM of Benzbromarone fore-aft motion Electric spike frequencies of administration Figure；

Fig. 4 B is that hippocampus of rats injects 400pA electric current 10 μM of Benzbromarone fore-aft motion Electric spike frequencies of administration Statistical chart；

Fig. 4 C is that hippocampus of rats 10 μM of Benzbromarone fore-aft motion current potentials of administration provide frequency diagram certainly；

Fig. 4 D is that resting membrane electric potential level statistic figure before and after 10 μM of Benzbromarones is administered in hippocampus of rats.

Fig. 5 shows in the embodiment of the present invention 4 Benzbromarone to the figure of mouse and rat inflammatory pain behavioral implications, in which:

Fig. 5 A is that Benzbromarone dose dependent decrease formalin inducing mouse inflammatory pain licks sufficient behavioral statistics figure；

Fig. 5 B is that Benzbromarone dose dependent weakens formalin inducing mouse inflammatory pain behavior marking statistical chart；

Fig. 5 C is that mouse administration 50mg/Kg Benzbromarone decrease bradykinin inducing mouse inflammatory pain licks sufficient behavioral statistics figure；

Fig. 5 D is that 50mg/Kg Benzbromarone decrease bradykinin inducing mouse inflammatory pain behavior marking statistical chart is administered in mouse；

Fig. 5 E is that 50mg/Kg Benzbromarone or/and 3mg/Kg XE991 is administered to formalin induced rat second in rat Phase inflammatory pain licks sufficient behavioral statistics figure；

Fig. 5 F is that 50mg/Kg Benzbromarone or/and 3mg/Kg XE991 is administered to formalin induced rat second in rat Phase inflammatory pain marking statistical chart.

Fig. 6 shows influence of the locally injecting Benzbromarone to rat inflammatory pain behavior in the embodiment of the present invention 4, in which:

Fig. 6 A is that ipsilateral local vola pedis injects the first phase of rat that Benzbromarone (100nmol/ plantar) induces formalin Inflammatory pain licks sufficient behavioral statistics figure；

Fig. 6 B is that ipsilateral local vola pedis injects Benzbromarone (100nmol/ plantar) to formalin induced rat the first phase inflammation Property beat soundly divide statistical chart；

Fig. 6 C is that ipsilateral local vola pedis injects the second phase of rat that Benzbromarone (100nmol/ plantar) induces formalin Inflammatory pain licks sufficient behavioral statistics figure；

Fig. 6 D is that ipsilateral local vola pedis injects Benzbromarone (100nmol/ plantar) to formalin induced rat the second phase inflammation Property beat soundly divide statistical chart；

Fig. 6 E is the first phase of rat that opposite side part vola pedis injects that Benzbromarone (100nmol/ plantar) induces formalin Inflammatory pain licks sufficient behavioral statistics figure；

Fig. 6 F is that opposite side part vola pedis injects Benzbromarone (100nmol/ plantar) to formalin induced rat the first phase inflammation Property beat soundly divide statistical chart；

Fig. 6 G is the second phase of rat that opposite side part vola pedis injects that Benzbromarone (100nmol/ plantar) induces formalin Inflammatory pain licks sufficient behavioral statistics figure；

Fig. 6 H is that opposite side part vola pedis injects Benzbromarone (100nmol/ plantar) to formalin induced rat the second phase inflammation Property beat soundly divide statistical chart.

Fig. 7 shows Benzbromarone in the embodiment of the present invention 5 and inhibits monosodium urate Salt treatment pain, in which:

Fig. 7 A is the time variation diagram that animal gait scores after solvent is injected intraperitoneally；

Fig. 7 B is the time variation diagram that animal gait scores after 10mg/Kg Indomethacin is injected intraperitoneally；

Fig. 7 C is the time variation diagram that animal gait scores after 50mg/Kg Benzbromarone is injected intraperitoneally.

Fig. 8 shows the mice convulsion effect that Benzbromarone induces maximal electroshock model in the embodiment of the present invention 6 Dose dependent figure.

Fig. 8 A be oral administration gavage various dose retigabine after to maximal electroshock stimulation induction convulsive attack protective rate Dose dependent figure；

Fig. 8 B is after various dose Benzbromarone is injected intraperitoneally to the convulsive attack protective rate of maximal electroshock stimulation induction Dose dependent figure.

Fig. 9 shows the figure that Benzbromarone influences the channel KCNQ4 and KCNQ5 channel current in the embodiment of the present invention 7, In:

Fig. 9 A is that current graph before and after 10 μM of Benzbromarones is administered in the channel KCNQ4 and the channel KCNQ5；

Fig. 9 B is that intensifying current multiple after 10 μM of Benzbromarones is administered in the channel KCNQ4 and the channel KCNQ5；

Fig. 9 C is that half activation voltage mobile range after 10 μM of Benzbromarones is administered in the channel KCNQ4 and the channel KCNQ5.

Specific embodiment

Below in conjunction with technical solution and the attached drawing specific embodiment that the present invention will be described in detail.

1 Benzbromarone of embodiment enhances the different aggressiveness channel current of KCNQ2 and KCNQ2/Q3

1.1 cell culture and transfection

Chinese hamster ovary cell (Chinese Hamster Ovary, CHO) (Cell Bank of Chinese Academy of Sciences) culture solution is matched Side: 50/50DMEM/F-12 (Gibco), 10% fetal calf serum (Fetal bovine serum, FBS) is added, and (Gibco, Australia are big Leah), 2mM L-Glutamine (Invitrogen).

To investigate influence of the Benzbromarone to the channel KCNQ2 and KCNQ2/Q3 of heterogenous expression, by liposome transfection side Method will be expressed with KCNQ2 and KCNQ3 gene plasmid in Chinese hamster ovary celI.

Specifically: expressing K CNQ2 homopolymer channel and the different aggressiveness channel KCNQ2/Q3, KCNQ2 plasmid are state by New York Professor D.Makinnon of university (State University of New York, Stony Brook) give, KCNQ3 plasmid It is given by professor M.C.Sanguinetti of University of Utah (University of Utah), Plasmid Base sequence is surveyed through gene Sequence simultaneously proved using Megalign 7 (DNAStar) sequence analyses and comparison software, respectively with the number announced in ncbi database For NM_133322.1 (KCNQ2), NM_004519.3 (KCNQ3) sequence is consistent.First 24 hours of transfection, uses trypsase It is laid in the ware of diameter 35mm after (Sigma, China) digestion.Transfection uses Lipofectamine2000^TMReagent (Invitrogen), it is operated according to its operating procedure (protocol) provided.24 hours after transfection, weight after cell dissociation It is newly laid on the slide of poly-L-Lysine (poly-L-lysine) (Sigma) immersion.Cotransfection GFP (green fluorescent protein), As the confirmation transfection cell under fluorescence microscope.

The different aggressiveness channel expressing K CNQ2/Q3 is transfected by plasmid mass ratio 50/50.

Electrophysiological recording on 1.2CHO cell

At room temperature, it is carried out using Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA) Full cell type voltage-clamp recording.Borosilicate glass capillary tube (World Precision Instrunents, Sarasota, FL it) is drawn into electrode, the resistance in electrode in tytosis after liquid is 3-5M Ω.

Intracellular fluid formula: 145mM KCl, 1mM MgCl₂, 5mM EGTA, 10mM HEPES, 5mM MgATP (KOH tune To pH=7.3).During record, held by BPS perfusion system (ALA Scientific Instruments, Westburg, NY) Continuous perfusion extracellular fluid.

Extracellular fluid formula: 140mM NaCl, 5mM KCl, 2mM CaCl₂, 1.5mM MgCl₂, 10mM HEPES, 10mM Glucose (NaOH is adjusted to pH=7.4).With DigiData 1322A in 9.2 software of pClamp after electric signal 2kHz filtering Digital signal is converted in (Molecular Devices, Sunnyvale, CA).Series resistance compensation 60-80%, electro physiology are ground Study carefully in recording process using multivoltage scheme.

1.3 Benzbromarones enhance the channel KCNQ2 and the different aggressiveness channel current of KCNQ2/Q3

Weigh 8.5mg Benzbromarone (Sigma company, analyze pure) be dissolved in 200 μ l DMSO (Sigma company, D5879, >= 99.5%) in, 100mM Benzbromarone mother liquor is obtained, and further obtains 20mM Benzbromarone storing liquid with DMSO gradient dilution, It is refrigerated in -20 DEG C.It takes 10 μ l in 20ml extracellular fluid in experimentation, and supplements 10 μ l DMSO to control to drug solns DMSO percent by volume is 1% (DMSO volume basis proportion is 1% in all test concentrations), mixes, reaches 10 μM of benzene bromines The grand test concentrations of horse.

In electrophysiological recording, the channel KCNQ2 and the different aggressiveness channel current record scheme of KCNQ2/Q3 are: Clamping voltages- 80mV, a series of 1500ms stimulation voltages (from -80mV to+50mV, being spaced 10mV) induced currents, by going through 1500ms-120mV Induce tail current.The channel KCNQ1 to be expressed electric current I under -10mV test voltage₀After stabilization, 10 μM are given using BPS perfusion system Benzbromarone, until electric current I stablizes.

1.4 experimental results statistical analysis

Electric physiological data analysis and utilization Clampfit 10.2 (Molecular Devices, Sunnyvale, CA), then benefit With GraphPad Prim 5 (GraphPad Software, San Diego, CA), voltage relies on activation curve and uses Boltzmann Equation (Boltzmann equation) fitting, dose-effect curve are fitted with Xi Er equation (Hill equation).Voltage according to Rely in activation curve, G is electric conductivity value, G_maxFor maximum conductance value, G/G_maxIt is fitted, is obtained using Boltzmann's equation after homogenization V_1/2Voltage, Δ V are activated for half_1/2Voltage is activated to move to left value for half, negative sign (-) indicates that current activation curve moves to left, positive sign (+) expression moves to right；I/I₀To activate multiple, I₀For preceding current value is administered, I is current value after administration.

As illustrated in fig. 1 and 2,10 μM of Benzbromarones are directly acted on into expression on the gonad cell of Chinese hamster The channel KCNQ2 (Fig. 1) and the different aggressiveness channel (Fig. 2) of KCNQ2/Q3, under -10mV test voltage, relative to (control before administration Group), channel current enhances 3.02 ± 0.12 (Fig. 1 .A) and 1.69 ± 0.16 times (Fig. 2 .A) respectively.Conductance voltage curve (G-V Curve) be response voltage gated ion channel degree of opening common counter, after 10 μM of Benzbromarones are administered, the channel KCNQ2 And the Δ V in the different aggressiveness channel KCNQ2/Q3_1/245.56 ± 4.50mV (Fig. 1 C) and 29.49 ± 4.46mV (Fig. 2 C) are moved to left respectively； Ion channel kinetics statistics shows that Benzbromarone significantly delays the shut-in time in the channel KCNQ2 and the different aggressiveness channel KCNQ2/Q3 normal Number τ_{It closes}(p < 0.0001) (Fig. 1 D and 1E, Fig. 2 B) shows that Benzbromarone can be by delaying channel to close and move to left channel conductance- Voltage relationship etc. and the effect for playing strengthening electric current intensity.Dose-dependent relationship is whether compound of reaction has pharmacology work One important indicator, as shown in Fig. 1 .B, Benzbromarone has the humidification of the channel KCNQ2 outward current apparent Dose-dependence, EC₅₀=4.68 ± 1.44 μM.Above data show Benzbromarone be a kind of novel channel KCNQ2 and The agonist in the different aggressiveness channel KCNQ2/Q3.

2 Benzbromarone of embodiment enhances endogenous KCNQ electric current in Dorsal Root Ganglion Neurons and inhibits action potential Provide frequency

In peripheral sensory nervous system, the outer peripheral portion of the Primary Sensory Neuron of impression and transmitting pain sensation information is known as pain Feel receptor, is the free nerve ending of no specialization on morphology.

After human body is by pain stimulus, the nerve impulse generated by these algesiroreceptor excitements can be through peripheral sensory mind Trident or Dorsal ganglion knot are reached through fiber, the dorsal horn of spinal cord is reached after changing member, is then reached greatly through certain Pathways Mediating Mesencephalic centre position, so as to cause nociception and pain.

Primary afferent neuron is responsible for the impression of body sensory system and is passed to, the part including four specializations: periphery mind Through tip, aixs cylinder, central terminals and cell space, research confirms there is the channel Kv7 in four parts of primary afferent neuron Expression.The cell space aggregation of primary afferent neuron constitutes dorsal root ganglion, and dorsal root ganglion neurons are widely used as ion channel The research object of agonist or inhibitor.Make to investigate Benzbromarone to the enhancing of endogenous KCNQ electric current in primary neuron With, and then action potential granting is adjusted, 10 μM of Benzbromarones are respectively acting on the rat of acute isolation by the present inventor Dorsal root ganglion neurons have studied Benzbromarone to endogenous in dorsal ganglion unit by voltage clamp and current clamp means The influence of KCNQ electric current and action potential.

The separation and culture of 2.1 dorsal ganglion units

Specific method is using dorsal ganglion unit enzymatic hydrolysis condition (Crozier RA etc., the J Neurosci being previously reported 2007,27:4492-4496.), acute isolation adult male SD rats dorsal root ganglion neurons cell, specific steps are as follows: take 150g or so SD male rat, after anesthesia, its canalis spinalis of exposure and intervertebral foramen under naked eyes.Vertebra is extracted one by one using microdissection tweezer Between dorsal root ganglion in hole, and remove the connection of its fiber as far as possible, be immediately placed on ice-cold oxygenation DMEM culture medium (Sigma, St.Louis, MO) in.After all extracing, collagenase digesting liquid (3mg/ml, type IA, Sigma) is moved into and with micro- Scissors shreds, and 37 DEG C digest 45 minutes, then digests again in tryptic digestive juice (2mg/ml, Type II-S, Sigma) 15 minutes.After, digestion is terminated with the DMEM culture medium containing 10% fetal calf serum, cell is blown and beaten repeatedly and extremely disperses, 40 μm After cell sieve filtration, suspension 800rpm is centrifuged 3 minutes.Liquid is discarded supernatant to connect after cell precipitate is resuspended with DMEM culture medium Kind, in advance on the coated coverslip of poly-D-lysine, is placed in 37 DEG C, 5%CO in 24 well culture plates₂It is cultivated in incubator.

The record of endogenous KCNQ electric current and action potential in 2.2 dorsal ganglion units

Dorsal ganglion unit endogenous KCNQ electric current is recorded as utilizing Axonpatch-700B amplifier (Molecular Devices, Sunnyvale, CA) carry out full cell type perforation voltage-clamp recording, endogenous KCNQ electric current Perforated patch clamp record Scheme is: liquid in the electrode of the amphotericin B containing 0.25mg/ml is charged in glass electrode.Cell is clamped down in -20mv, super Command potential level is returned after changing to -50mv, records endogenous KCNQ current amplitude at this time.Dorsal ganglion unit action potential It is recorded as carrying out full cell type electricity using Axonpatch-700B amplifier (Molecular Devices, Sunnyvale, CA) Stream pincers record, dorsal ganglion unit action potential record scheme is: cell being clamped down in 0pA, to endocellular injection 400pA, is continued The depolarization current of time 1s stimulates, operation of recording potential amplitude.Electrode enters 2-3 megaohms of water resistance, series resistance compensation 60- 90%, record condition temperature is 20-22 DEG C.

Intracellular fluid formula: 80mM K acetate, 30mM KCl, 1mM CaCl₂, 3mM MgCl₂, 3mM EGTA, 40mM HEPES (KOH is adjusted to pH=7.4)；

Extracellular fluid formula: 144mM NaCl, 2.5mM KCl, 2mM CaCl₂, 0.5mM MgCl₂, 5mM HEPES, 10mM Glucose (NaOH is adjusted to pH=7.4).

2.3 Benzbromarones enhance dorsal ganglion unit endogenous KCNQ electric current and inhibit action potential granting

It weighs respectively Benzbromarone (Sigma company analyzes pure), XE991 (endogenous KCNQ current selective inhibitor, Sigma company analyzes pure) it is dissolved in extracellular fluid to 10 μM of test concentrations.

It perforates under voltage-clamp recording mode in full cell type, by dorsal ganglion unit in -20mV stimulation voltage, perfusion is extracellular Stable current amplitude is defined as I0 when liquid, and current amplitude at 10 μM of perfusion is defined as I, last perfusion endogenous KCNQ electric current Inhibitor XE991, confirmation record current are endogenous KCNQ electric current.It perforates under voltage-clamp recording mode in full cell type, statistics Dorsal ganglion unit is before and after 10 μM of Benzbromarones of perfusion, and the action potential number that injection 400pA electric current is induced is to determine benzene Influence of the bromine Ma Long to dorsal ganglion unit action potential granting (nerve excitability).

2.4 interpretation of result

The experimental results showed that 10 μM of Benzbromarones significantly increase dorsal ganglion unit endogenous KCNQ electric current, relative to administration Before (control group), 1.98 ± 0.19 times of intensifying current (p < 0.001) (Fig. 3 A and 3B) this humidifications can be endogenous by 10 μM Property KCNQ current selective inhibitor XE991 cancel, relative to be administered before 0.49 ± 0.05 times of current reduction (Fig. 3 A and 3B), table Bright Benzbromarone to the humidification of dorsal ganglion unit outward current is reached by enhancing endogenous KCNQ electric current.Endogenous KCNQ electric current is a kind of potassium current of time and voltage-dependent, slowly activation and de-inactivation, non-inactivation in neuron, Braking action is played in the electric discharge repeatedly of neuron action potential, enhancing endogenous KCNQ electric current can inhibit neuron action electric The granting of position.In Dorsal root ganglion, Benzbromarone can reduce neuron resting potential level and significantly inhibit neuron The granting frequency of action potential.After 10 μM of Benzbromarones are administered, the same electric current for injecting 400pA duration 1s is induced Dorsal root ganglion action potential number, by be administered first 18.67 ± 6.67 be reduced to administration after 2.33 ± 0.76 (p < 0.05) (Fig. 3 C and 3D).Influence of the Benzbromarone to action potential can be by 10 μM of endogenous KCNQ current selective inhibitor XE991 cancels, and it is 14.6 ± 2.08 (Fig. 3 C and 3D) that action potential frequency, which reverses,.The above experimental data shows Benzbromarone Enhance endogenous KCNQ electric current in dorsal root ganglion neurons and dorsal root ganglion neurons action potential is inhibited to provide frequency.

3 Benzbromarone of embodiment enhances endogenous KCNQ electric current in hippocampus of rats and action potential is inhibited to provide frequency Rate

Epileptic condition is a kind of caused maladjusted nervous system disease of cerebral neuron paroxysmal abnormality electric discharge, shows as transporting Dynamic, feeling, consciousness, spirit, vegetative nerve etc. obstacle.Since hippocampal formation and functional study are relatively clear, hippocampal pyramidal Neuron is the common object of antiepileptic electro physiology detection.

The separation and culture of 3.1 hippocampal neurons

The newborn 24 hours pairs of hippocampus of SD rat of acute isolation, thoroughly removed in the phosphate buffer of ice bath meninx and Cerebral microvascular moves into trypsase (2.5mg/ml, Type II-S, Sigma), and micro- scissors shreds, and 37 DEG C digest 30 points Clock.After, with isometric growth medium (penicillin and streptomysin, 2mM glutamine containing 10% fetal calf serum, 1% DMEM/F12 digestion) is terminated, blows and beats cell repeatedly to dispersion, after 70 μm of cell sieve filtrations, suspension 800rpm is centrifuged 3 minutes. Liquid is discarded supernatant, after cell precipitate is resuspended with growth medium, by 100,000 every milliliter of final concentration of cells, is inoculated in the training of 24 holes It supports in plate in advance on the coated coverslip of poly-D-lysine, is placed in 37 DEG C, 5%CO₂It is cultivated in incubator.Culture 6 hours Afterwards, (1% penicillin and chain is added in the Neurobasal A that training liquid all changes 2%B-27 and 0.5mM 1%GlutaMAX into Mycin), change half culture medium within every 3 days.After culture 12~15 days, electrophysiological recording endogenous KCNQ electric current.

The record of 3.2 hippocampal pyramidal neurons resting membrane electric potential levels and action potential

Hippocampal neuron endogenous KCNQ electric current records inside and outside formula of liquid:

Intracellular fluid formula: 140mM KCl, 1mM MgCl₂, 1mM CaCl₂, 20mM EGTA, 10mM HEPES (KOH tune To pH=7.3)；

Extracellular fluid formula: 135mM NaCl, 5mM KCl, 2mM MgCl₂, 10mM HEPES, 10mM glucose, 0.001mM tetraodotoxin (NaOH is adjusted to pH=7.3).

In electrophysiological recording, the neuron for selecting cell space form tapered is recorded, register instrument, electric current and voltage thorn Swash scheme and the same dorsal root ganglion neurons of dosage regimen.3.3 interpretation of result

The experimental results showed that 10 μM of Benzbromarones significantly reduce hippocampal pyramidal neurons resting potential level and significantly inhibit The granting frequency of neuron action potential.After 10 μM of Benzbromarones are administered, the same electric current for injecting 400pA duration 1s, institute The hippocampal pyramidal neurons action potential number of induction, by be administered first 11.33 ± 2.27 be reduced to administration after 0.33 ± 0.41 A (p < 0.05) (Fig. 4 A and 4B), action potential restores to 3.33 ± 1.47 after eluting withdrawal.It is dynamic in hippocampal pyramidal neurons Make current potential from providing in experiment, 10 μM of Benzbromarones reduce hippocampal pyramidal neurons resting membrane electric potential level and weaken action potential It provides frequency (Fig. 4 C), resting membrane electric potential is reduced to -69.64 ± 2.22mV after administration by -61.45 ± 1.37mV before being administered (p < 0.05) (Fig. 4 D).The above experimental data shows that Benzbromarone reduces hippocampal pyramidal neurons level of membrane potential and movement electricity Provide frequency in position.

4 Benzbromarone of embodiment alleviates the mouse inflammatory pain behavior induced by bradykinin and the rat induced by formalin With mouse inflammatory pain behavior

Above-mentioned experimental data shows that Benzbromarone can pass through enhancing endogenous KCNQ electric current and weaken primary neuron movement The granting of current potential implies that Benzbromarone can be used for treating the pain as caused by peripheral inflammation or neurotrosis.

The foundation of the pain model of 4.1 inductions

From Dubuisson and Dennis by the way that 4% formalin, the formal of foundation is subcutaneously injected to rat hindleg metacarpus Woods model is the ideal model of generally acknowledged aseptic inflammatory pain, certain features of model analog people's pain after injury, pain Behavior is similar and reproducible to clinical manifestation (Pain 1977,4 such as Dubuisson D (2), 161-74).Formalin induction Inflammatory pain behavior according to the pain reaction of animal, the strong of 0~9 minute acute stage (the first phase) and 10~60 minute can be divided into The straight phase (the second phase), the first phase is directly algesiroreceptor to be stimulated to cause by formalin, and the second phase is caused by formalin Inflammatory reaction and central sensitization effect cause.

In this zoopery, 0.9% life that 100 μ l, 37%~40% formalin solution is diluted in 900 μ l is drawn It manages in salt water, obtains about 4% formalin proinflammatory agent.It draws 30 μ l and is injected in SD rat/Sprague-Dawley rat (SD rat and KM mouse are mentioned by Shanghai Slac Experimental Animal Co., Ltd. for (150~180g) and KM mouse/kunming mice For) left back vola pedis it is subcutaneous, observe and record the ANIMAL PAIN behavior reaction in injection of formalin proinflammatory agent 1h.Pain reaction To lick sufficient time and marking as standard, criterion of giving a mark are as follows: lick 3 points/time of foot, tremble 2 points/time of foot, lift 1 point/time of foot.

4.2 Benzbromarones alleviate the Pain behaviour reaction of formalin induction

The Benzbromarone (Sigma company analyzes pure) for weighing 15mg, 25mg and 40mg respectively is dissolved completely in 500 μ l In DMSO (Sigma company, D5879, >=99.5%), the Tween-80 that 9.5ml 1% is added (takes 1ml Tween-80 to be dissolved in 99ml ultrapure water), obtain the grand injection of bromine horse of 1.5mg/ml, 2.5mg/ml and 4mg/ml.Injection of formalin proinflammatory agent Benzbromarone injection (the whole 5%DMSO+ of three kinds of concentration is injected respectively in the right abdominal cavity of KM mouse by 0.2ml/10g before 30min 95% 1%Tween-80), obtain three kinds of various doses mouse Benzbromarone dosage (30mg/Kg, 50mg/Kg and 80mg/Kg)。

Weigh 125mg Benzbromarone (Sigma company, analyze pure) be dissolved in 500 μ l DMSO (Sigma company, D5879, >= 99.5%) in, dissolution completely, is added the Tween-80 (1ml Tween-80 is taken to be dissolved in 99ml ultrapure water) of 9.5ml 1%, obtains 12.5mg/ml Benzbromarone injection.It is infused by 0.4ml/100g in SD Rat Right abdominal cavity before injection of formalin proinflammatory agent 30min Penetrate (Intraperitoneal injection, writes a Chinese character in simplified form: i.p.) Benzbromarone injection (1% of whole 5%DMSO+95% Tween-80), the rat Benzbromarone dosage of 50mg/Kg is obtained.

Enhancing endogenous KCNQ electric current generation is acted through to investigate Benzbromarone behavior of relieving pain in animal level, In SD rats by intraperitoneal injection XE991 and XE991+ Benzbromarone, record observation endogenous KCNQ electric current specific inhibitor XE991 Influence to Benzbromarone analgesic activity.

Weigh 9mg XE991 (Sigma company, analyze pure) be dissolved in 300 μ l DMSO (Sigma company, D5879, >= 99.5%) in, dissolution completely, takes 0.25ml (to take 1ml Tween-80 to be dissolved in 99ml ultrapure in the Tween-80 of 4.75ml 1% Water), obtain 1.5mg/ml XE991 injection.By 0.4ml/100g in Rat Right abdomen before injection of formalin proinflammatory agent 30min Chamber injects XE991 injection (1%Tween-80 of whole 5%DMSO+95%).

Rat and mouse inflammatory pain behavior record: in 60 minutes after vola pedis injection of formalin, record rat lick foot/ Sting the sufficient time (unit: s/ seconds)；And Pain behaviour number is recorded according to scoring criterion.

As shown in Figure 5 A and 5B, mouse inflammatory pain row caused by the dose-dependent alleviation formalin of Benzbromarone energy For.30mg/Kg, the Benzbromarone of 50mg/Kg, 85mg/Kg is administered, mouse licks the sufficient time relative to control group (not to benzene bromine Grand group of horse) 215.27 ± 43.59s be reduced to respectively 142.00 ± 38.27s, 66.36 ± 21.97s (p < 0.05), 59.44 ± 13.19s(p<0.05)；The Pain behaviour of mouse is given a mark is reduced to 147.57 relative to the 189.00 ± 31.43 of control group respectively ±26.35,80.64±21.16(p<0.05),51.11±11.68(p<0.01).Above data shows that Benzbromarone can be alleviated The inflammatory pain as caused by formalin.In the rat inflammatory pain model of formalin induction, 50mg/Kg Benzbromarone is significantly slow ANIMAL PAIN behavior reaction is solved, while the KCNQ channel selectivity inhibitor XE991 that 3mg/Kg is injected intraperitoneally reverses Benzbromarone Analgesic activity (Fig. 5 E and 5F), and individually inject XE991 can not alleviate formalin induction inflammatory pain, illustrate benzene bromine The grand analgesia of horse is reached by enhancing KCNQ channel function.

The foundation of the pain model of 4.3 bradykinins induction

One most evident characteristic of algesiroreceptor is exactly can be by institute in the case of damaged tissues, inflammation and metabolic stress The inflammatory mediator institute enhanced sensitivity of release, wherein these inflammatory mediators include: bradykinin, it is nerve growth factor, ATP, preceding Column parathyrine and proton etc..In participating in numerous inflammatory mediators that inflammatory pain generates, bradykinin is wherein important A kind of inflammatory mediator.It is by kininogen when body is stimulated by pathology such as tissue damage, anoxic, inflammation, in kassinin kinin It is transformed under the action of release enzyme.Bradykinin effect is very extensive, it makees in addition to participating in smooth muscle contraction, glandular secretion etc. With outer, moreover it is possible to which sensitizing sensory nerve member simultaneously causes inflammatory reaction, is especially of great significance during the pain sensation generates.Inflammatory Bitterly, rheumatoid pain and the pain sensation caused by anoxic are all with the raising of bradykinin concentration.

It is acted in the inflammatory pain model that bradykinin induces to investigate Benzbromarone, weighs 3mg bradykinin and be dissolved in 150 μ l 0.9% physiological saline in, obtain the bradykinin proinflammatory agent of 20 μ g/ μ l.It draws 10 μ l and is injected in KM mouse/kunming mice The left back vola pedis of (18~22g) (KM mouse is provided by Shanghai Slac Experimental Animal Co., Ltd.) is subcutaneous, observes and remembers Record the ANIMAL PAIN behavior reaction in injection of formalin bradykinin proinflammatory agent 30min.Pain reaction is to lick sufficient time and lift foot Time and marking are used as standard, criterion of giving a mark are as follows: lick 2 points/time of foot, lift 1 point/time of foot.

4.4 Benzbromarones alleviate the inflammatory pain of bradykinin induction

Weigh 15mg Benzbromarone (Sigma company, analyze pure) be completely dissolved in 300 μ l DMSO (Sigma company, D5879, >=99.5%) it in, takes 0.25ml in the Tween-80 (1ml Tween-80 is taken to be dissolved in 99ml ultrapure water) of 4.75ml 1%, obtains 2.5mg/ml Benzbromarone injection.It is infused by 0.2ml/10g in the right abdominal cavity of mouse before vola pedis injects bradykinin proinflammatory agent 30min Penetrate Benzbromarone injection (1%Tween-80 of whole 5%DMSO+95%).

As seen in figs. 5c and 5d, after 50mg/Kg Benzbromarone being administered, the lift of mouse is sufficient and licks the sufficient time by relative to control 270.67 ± 35.00s of group (not giving Benzbromarone group) is reduced to 67.00 ± 11.00s (p < 0.001)；Pain behaviour marking By being reduced to 39.00 ± 6.00 (p < 0.01) relative to the 67.00 ± 5.00 of control group.Above data shows Benzbromarone energy Alleviate the inflammatory pain as caused by bradykinin.

4.5 Benzbromarone local administrations alleviate the Pain behaviour reaction of formalin induction

Apply influence of the Benzbromarone to Pain behaviour to investigate part, 100nmol/ plantar is subcutaneously injected in SD rat paw Benzbromarone (the Benzbromarone injection that 50 μ l concentration are 2mmol/L), injects the good fortune of 30 μ l 4% in same position after five minutes That Malin's proinflammatory agent (same to side injection), observes and records the ANIMAL PAIN behavior reaction in injection of formalin 60min.Pain is anti- It should be to lick the sufficient time and lift sufficient time and marking as standard, marking criterion are as follows: lick 3 points/time of foot, tremble 2 points/time of foot, lift foot 1 Point/time.

Weigh 10mg Benzbromarone (Sigma company, analyze pure) be completely dissolved in 590 μ l DMSO (Sigma company, D5879, >=99.5%) it in, takes 0.5ml in the Tween-80 (1ml Tween-80 is taken to be dissolved in 99ml ultrapure water) of 9.5ml 1%, obtains The Benzbromarone locally injecting liquid of 2mmol/L.

Ipsilateral vola pedis locally injecting 100nmol/ plantar Benzbromarone, significant the first phase and second for alleviating Formalin induction Phase inflammatory pain (Fig. 6 A-D).Due to tissue fluid venous return and blood circulation, vola pedis subcutaneous injection Benzbromarone may include 100nmol/ plantar Benzbromarone is subcutaneously injected in the left back vola pedis of SD rat thus, after five minutes in the right side in the centre effect of Benzbromarone The formalin proinflammatory agent (to side injection) of 30 μ l 4% of metapedes plantar subcutaneous injection, record observation 1h, to investigate locally injecting benzene Bromine Ma Long influences (Fig. 6 E-H) to the Pain behaviour that opposite side vola pedis formalin induces.

The experimental results showed that locally injecting Benzbromarone significantly alleviates the rat pain row with the induction of position formalin For, and the rat Pain behaviour that locally injecting Benzbromarone induces opposite side formalin illustrates locally injecting benzene bromine without influence Horse is grand significantly to relieve pain, and this pain relief effect mainly passes through peripheral neverous system and works.

5 Benzbromarone of embodiment alleviates the acute arthritis pain of monosodium urate salt crystal induction

It is now recognized that Benzbromarone urinates clinical application as uricosuric, treatment gout effect, which mainly passes through, inhibits kidney small Pipe reduces Plasma Uric Acid content to the reabsorption of uric acid, restores serum uric acid level to stable state (Rider TG etc. Rheumatology(Oxford).2010,49(1):5-14).Above-mentioned experiment shows that Benzbromarone can be by enhancing the channel KCNQ Function plays analgesic activity.It is acted on to investigate Benzbromarone to gouty arthritis pain, establishes the acute of monosodium urate Salt treatment Gouty arthritis model.

The foundation of the experimental study model of 5.1 monosodium urate Salt treatments

5.1.1 the preparation of monosodium urate salt

Uric acid preparation method was utilized in 1994 using Roberge et al., specifically: weighing 1.0084g uric acid crystal (Sigma) and 200ml deionized water is added in 0.24g sodium hydroxide (Chinese medicines group chemical reagents corporation), boils dissolution, uses 1mol/L sodium hydroxide tune pH value is to 7.5.0.96g sodium chloride crystal is added, to solving completely, room temperature is cooling, obtains needle-shaped for stirring Monosodium urate salt crystal (Monosodium urate crystals；MSU), kept dry.

5.1.2 the foundation of the pain model of monosodium urate Salt treatment

It is closed using Coderre et al. in the acute gout in rat ankle joint injection monosodium urate salt crystal induction in 1987 Scorching model is saved, specifically: 100mg monosodium urate salt crystal is weighed, is dissolved in the phosphate buffer of 4ml, oscillation is dissolved completely, Obtain the monosodium urate salt suspension of 25mg/ml.It is breathed and is anaesthetized using isoflurane, anesthesia SD rat/Sprague-Dawley is big After mouse (180~220g) (SD rat is provided by Shanghai Slac Experimental Animal Co., Ltd.), closed in rat left hind ankle The monosodium urate salt suspension that 50 μ l concentration are 25mg/ml is injected at section (for 1.25mg/ ankle-joint).After injecting 22h, observation is big Mouse gait evaluates pain degree, standards of grading: 0 point is normal gait, and biped uniformly lands；1 point is walked lamely to be slight, and left hind omits Have completely；2 points are walked lamely for moderate, and left hind just touches ground；3 points are walked lamely for severe, and left hind leaves ground completely, tripodia Ground walking.In addition, it is 0.5 point, 1.5 points and 2.5 points that the gait behavior to fall between, which is successively given a mark,.

5.2 Benzbromarones alleviate the Pain behaviour reaction of monosodium urate Salt treatment

To investigate effect of the Benzbromarone to the rat acute arthritis ache behavior of monosodium urate Salt treatment, ache establishing After pain model 22h, record pain marking, and made respectively with the anti-inflammatory analgesic Indomethacin of 10mg/Kg and solvent (control group) For positive control and negative control.

It weighs 25mg anti-inflammatory analgesic and wants Indomethacin (Indomethacin；Sigma), it is dissolved completely in 0.5ml DMSO, The Tween-80 (1ml Tween-80 is taken to be dissolved in 99ml ultrapure water) of 9.5ml 1% is added, obtains 2.5mg/ml Indomethacin note Penetrate liquid.

The pain model that the Benzbromarone injection and injection system of 12.5mg/ml is induced with formalin.

According to 0.4ml/100g standard abdomen difference chamber injection Indomethacin, solvent and Benzbromarone, after 1h, 2h and 3h is administered The Pain behaviour degree of animal is observed and recorded respectively.

As shown in Figure 7 A, intraperitoneal injection solvent (control group) Pain behaviour of animal in the 3h of record is unchanged, explanation Injection itself and solvent are on ANIMAL PAIN behavior without influence.Significantly alleviate ankle-joint list after intraperitoneal administration 10mg/Kg Indomethacin The rat acute gouty arthritis pain reaction (p < 0.0001) of natriuresis hydrochlorate induction, the above result shows that, the pain journey of animal Degree mitigation is the effect (Fig. 7 B) of drug.After the Benzbromarone of intraperitoneal administration 50mg/Kg, the pain degree of animal significantly mitigates (p < 0.0001), illustrate that Benzbromarone can alleviate the Acute Pain (Fig. 7 C) of monosodium urate Salt treatment.

Protective effect of 6 Benzbromarone of embodiment to MES (maximal electroshock) the mice convulsion model induced

For the antiseizure for detecting Benzbromarone in whole animal level, inventor is using maximal electroshock induction Mice convulsion model inspection protective rate of the various dose Benzbromarone to convulsive attack.Male KM mouse (weight is 22 ± 2g) 90 stimulation voltage 160V, wave number (i.e. stimulation times are arranged using configuration 8 using YLS-9A type physiological and pharmacological electronic stimulation instrument 5.4s).Ear-clip electrodes give stimulation 1 time, stretch so that hind leg is mandatory as convulsions index, experiment the previous day is screened.Experiment When, the Benzbromarone of (0mg/Kg, 30mg/Kg, 50mg/Kg, 85mg/Kg and 100mg/Kg) is injected intraperitoneally within 30 minutes in advance, so MES test is carried out according to the parameter of the previous day setting afterwards, observes and records the response situation of animal immediately.In experiment, using filling The retigabine of stomach (0mg/Kg, 3mg/Kg, 10mg/Kg, 30mg/Kg and 60mg/Kg) is as positive reference compound (Fig. 8 A).

Experimental results are shown in figure 8, and dose-dependence, intraperitoneal injection is presented in the anti-epileptic protective rate of Benzbromarone The anti-seizure protective rate of 85mg/Kg Benzbromarone reaches 80%, and the anti-epileptic of 100mg/Kg Benzbromarone is injected intraperitoneally The protective rate of breaking-out reaches 100% (Fig. 8 B), illustrates that Benzbromarone has anti-epileptic effect.

7 Benzbromarone of embodiment enhances KCNQ4 channel current

KCNQ4 is predominantly located in inner ear, participates in the formation of the sense of hearing, and miopragia after mutation causes non-syndrome often to contaminate Colour solid dominant heredity is deaf (Nonsyndromic Sensorineural Deafness type 2, DFNA2).Furthermore The channel KCNQ4 and KCNQ5 is also distributed in smooth muscle cell, by influencing cell membrane potential level, adjusts relaxing for smooth muscle It opens and shrinks.Therefore, the channel KCNQ4 and the channel KCNQ5 are the important drugs target spot of deaf and smooth muscle conditions treatment, KCNQ4 Channel and KCNQ5 channel agonist can be used for deaf and smooth muscle conditions therapeutic agents.

KCNQ4 plasmid and KCNQ5 plasmid are respectively by Texas health science center (University of Texas Health Science Center, San Antonio) M.Shapiro professor and Loyola University Chicago (Loyola University Chicago) Kenneth professor Byron give, sequence through gene sequencing and utilize Megalign 7 (DNAStar) sequence analyses and comparison software proves, be with the number announced in ncbi database NM_004700.3 (KCNQ4) and NM_019842.3 sequence is consistent, and expression, record and the data analysing method step in channel are the same as embodiment 1.

Under -10mV test voltage, relative to the channel (control group) KCNQ4 and KCNQ5 electricity before 10 μM of Benzbromarones of administration Stream enhances 1.88 ± 0.11 times and 3.11 ± 0.54 times (Fig. 9 A and 9B), Δ V respectively_1/2Move to left 24.76 ± 3.28mV and 25.48 ± 1.27mV (Fig. 9 C).Show that Benzbromarone can be used for using KCNQ4 and KCNQ5 as the treating correlative diseases of target spot and/or prevention, Such as hypertension, antenatal throe smooth muscle conditions and non-syndrome autosomal dominant deafness auditory system disease.

Claims (3)

1. a kind of Benzbromarone, for treating epilepsy, treats non-syndrome autosomal dominant deafness disease in preparation Application in disease, and/or the drug for the treatment of inflammatory pain.

2. application as described in claim 1, wherein Benzbromarone is as KCNQ agonist.

3. application as claimed in claim 2, wherein the KCNQ agonist be KCNQ2 agonist, KCNQ2/Q3 agonist, KCNQ4 agonist, KCNQ5 agonist or endogenous KCNQ electric current agonist.