CN101199521A - Application for preparing therapy antiarrhythmic medicament of Zacopride - Google Patents

Abstract

The invention relates to the medicine field, particularly relating to a drug for treating arrhythmia, in particular to an application of Zacopride in preparing a drug for treating the arrhythmia. The invention aims at solving the problems that the present antiarrhythmic drugs have the effect of causing arrhythmia, and IK1 selective agonist has not been discovered at home and abroad so far. The Zacopride is an IK1 selective agonist which is discovered at present for the first time and is used on an antiarrhythmic drug. Used as a drug for treating ischemia-reperfusion arrhythmia, the Zacopride has the similar effect to that of lidocaine, takes effect stably and does not obviously affect the rhythm with wide safety range. In the experiments of arrhythmia induced by ischemia-reperfusion, lidocaine can make the rhythm slower, while the Zacopride has no obvious effect; and when the dosage of the lidocaine is increased by one-third, the antiarrhythmic effect is obviously weakened, while such phenomenon occurs when the Zacopride reaches one hundred times of effective concentration.

Description

Bundle is examined the application of Billy as preparation treatment antiarrhythmic medicament

Technical field

The present invention relates to field of medicaments, relate generally to the ARR medicine of treatment, be specially to prick and examine the application of Billy as preparation treatment antiarrhythmic medicament.

Background technology

Arrhythmia is meant the unusual of the palmic rate and the rhythm and pace of moving things, and arrhythmia is common clinically cardiovascular disease, and severe arrhythmia is patient's one of underlying cause of death often.

Myocardial ischemia, anoxia, acidosis/alkalosis, electrolyte disturbance, drug intoxication, myocardial damage etc. all can bring out arrhythmia.The fundamental mechanism that arrhythmia takes place is turned back, self-disciplining is unusual and trigger sexual activity.That is all arrhythmia all are because conduction of impulse obstacle and impulsion form obstacle or the two has concurrently caused.The mechanism that forms severe arrhythmia is very complicated; often not single mechanism, but search to the bottom that the active change of ion channel function is all arranged; cause that thus the variation of ion flow can influence the generation and the form of action potential, thereby have proarrhythmia or control ARR effect.

The mechanism of action of present used anti-arrhythmic:

1. reduction self-disciplining

Increase maximum diastolic potential (4 phase), promote 4 phase K ⁺Outflow.

The 4 very fast degree that are divided by that slow down suppress Na ⁺, Ca ²⁺Interior stream.

2. elimination after-depolarization, triggered activity

After-depolarization early: calcium antagonists.

Delayed afterdepolarization: calcium antagonists, blocking-up Na ⁺, Ca ²⁺Exchanging electric current.

3. the change membrane responsiveness changes conduction, and elimination is turned back

The reinforcing membrane reactivity increases transmembrane potential, and the transmembrane potential absolute value is big more, and reactivity is high more, then can change conduction, eliminates unidirectional block (phenytoin Sodium).

Reduce membrane responsiveness, the conduction of slowing down causes region of unidirectional block to become two-way retardance fully, eliminates the loop of turning back.

4. change action potential duration, APD and effective refractory period, elimination is turned back

The absolute effective refractory period that prolongs: over reach current potential time-histories, prolong effective refractory period, elimination is turned back.

Prolong effective refractory period relatively: the shortening of action potential duration, APD is greater than the shortening of effective refractory period, and elimination is turned back.

Make the homogenization of the inhomogenous effective refractory period trend of flanking cell.

For a long time, people are devoted to the research of antiarrhythmic drug always.The antiarrhythmic drug significant feature target spot of clinical practice at present is voltage-gated sodium channel (I _Na), Delayed Rectifier Potassium Channels (I _K) and L type calcium channel (I _Ca-L), according to the effect of medicine, will treat the tachyarrhythmia medicine and be divided into four classes ion channel and receptor:

I class: sodium channel blocade

I _a: moderate strength (appropriateness retardance sodium channel), as quinidine, procainamide

I _b: slightly block the sodium channel, as lignocaine, phenytoin Sodium, mexiletine, tocainide

I _c: severe retardance sodium channel, as Propafenone, flecainide

This type of medicine can make the blocking-up sodium channel, changes the conduction in unidirectional tissue district, and elimination is turned back.

The II class: the beta receptor blocking agent, as Propranolol, this type of medicine can make 4 phase depolarization currents reduce, and self-disciplining reduces.

The III class: over reach current potential time-histories medicine (potassium channel blocade), as amiodarone, suppress I _K, repolarization 3 phase efflux of K+ ions are reduced, over reach current potential time-histories and effective refractory period prolong, and elimination is turned back.

The IV class: calcium channel blocker, as verapamil, the blocking-up calcium channel reduces action potential 4 phase flow of calcium ions, and the slow reacting cell self-disciplining reduces, and can be used to treat supraventricular arrhythmia.

---above four classes are mainly treated tachyarrhythmia

---to slow arrhythmia, available atropine, isoproterenol are treated

---other drug: can treat supraventricular tachycardia as phenylephrine, neostigmine

No matter which kind of antiarrhythmic drug all has certain arrhythmogenic effect at present.I class medicine can slow down to conduct to bring out and turn back because of the blocking-up sodium channel; III class medicine brings out long QT syndrome because of over reach current potential time-histories.This has caused people's attention, and it is extremely urgent to seek new safer Therapeutic Method.After middle nineteen nineties, the cardiac catheter interventional technique is treated all kinds of arrhythmia and has been obtained remarkable progress, has opened up the new direction for the treatment of irregular heart pulse.But interventional therapy has certain indication scope, so for most patients with arrhythmia, Drug therapy is still the main method of treatment or essential ingredient, wherein lignocaine is the medicine of therapeutic room's arrhythmia of generally acknowledging.

During the former studies antiarrhythmic medicament, inward rectifyimg potassium channel (I _K1Passage) is a target spot that gets the brush-off.Because all antiarrhythmic drugs all are channel blockers so far, and I _K1Be myocardium topmost background outward current, blocking-up I _K1But though passage over reach current potential time-histories, simultaneously owing to the background outward current reduce cause resting potential to reduce or the maximal diastolic potential of autorhythmic cell reduces, thereby reduced myocardium conductivity, improved self-disciplining, produce arrhythogenic effect.Therefore also there is not a kind of specific inhibition I so far _K1Antiarrhythmic drug come out.Kubo cloned successful I in 1993 _K1Since the passage, to I _K1Structure, function and electrophysiological characteristics obtained more deep research.Studies show that the main diseases of Andersen-Tawil syndrome is because of being KCNJ gene (ventricular muscles I _K1The major gene of passage) sudden change causes the Kir2.1 channel function to weaken or loses; Studies show that of computer simulation, the electric conductance of reduction Kir2.1 passage, the actuatable current potential multipole of doing postpones at the end eventually, and resting potential reduces (depolarization), spontaneous ventricular arrhythmia occurs; Zoopery confirms that transmembrane potential reduces and ARR generation the arrhythmogenic effect of Endothelin, Angiotensin II, Ca in the born of the same parents during heart failure during myocardial ischemia ²⁺Equal and I such as the arrhythmia due to concentration raises _K1The inhibition of electric current is relevant.Therefore it is contemplated that exciting I _K1Passage will have the antiarrhythmic effect.But yet there are no selectivity I so far both at home and abroad _K1The report of channel agonist is not more seen by the exciting I of specificity _K1Passage is brought into play the report of arrhythmia effect the human or animal.

Summary of the invention

The present invention all has certain arrhythmogenic effect in order to solve existing antiarrhythmic drug, and does not find I both at home and abroad as yet so far _K1The problem of selective agonist provides and pricks the new purposes of examining Billy (Zacopride), i.e. new application in pharmacy---and prick and examine the application of Billy as preparation treatment antiarrhythmic medicament.

In fact, the present invention relates to prick and examine the application of Billy as preparation treatment antiarrhythmic medicament.

It is capsule, microcapsule, liposome, granule, injection, tablet and oral liquid that described bundle is examined Billy's preparation.

Bundle is examined Billy (Zacopride), and gastric motility promoter is croak pyridine benzamide derivant, belongs to 5-HT ₃Receptor antagonist and 5-HT ₄Receptor stimulating agent is used to promote the running of gastric motility and intestinal at present, also as promoting brain metabolic cycles medicine treatment senile dementia.The chemical structural formula that bundle is examined Billy is:

In the examination test of a short intestinal power medicine arrhythmogenic effect, we find to prick and examine the no proarrhythmia side effect of Billy (Zacopride), and can strengthen I _K1Channel current, but to I _Ca-L, I _Na, I _To, I _K, I _Na/CaAll do not make significant difference etc. main myocardium ion current.This results suggest bundle is examined Billy (Zacopride) and be can be used as I _K1Selective agonist becomes a kind of new antiarrhythmic drug.

The present invention adopts SD rat and Cavia porcellus (only to be used to observe I _KPassage) experimentizing, examining Billy (Zacopride) to the influence of ventricular muscle cell ion channel current and measure to prick and examine Billy (Zacopride) to rat ischemia and the influence of pouring into the arrhythmia effect again, confirmed above-mentioned enlightenment by measure pricking.The concrete confirmation thes contents are as follows:

A.1～30 in the μ mol/L scope, bundle is examined Billy (Zacopride) and is concentration dependent enhancing I _K1, 10 μ mol/L bundle is examined Billy (Zacopride) makes I _K1Electric current density increases by 29.45% (P＜0.01).

1～30 μ mol/L scope Nei Zhakao Billy (Zacopride) is to I _Ca-L, I _Na, I _To, I _K, I _Na/CaDo not have obviously influence, show to prick and examine the specificity of Billy (Zacopride) the passage agonism.

Bundle examine Billy (Zacopride) to resting membrane electric potential, action potential amplitude (Action potential Amplitude, APA), action potential multipole 50% time-histories (Action potential duration at 50%repolarization, APD ₅₀) and action potential multipole 90% time-histories (Action potential duration at 90%repolarization, APD ₉₀) influence.In 3～40 μ mol/L scopes, prick and examine Billy (Zacopride) but concentration dependent increase resting potential, increase action potential amplitude, current potential multipole 50% time-histories (APD under reach ₅₀) and action potential multipole 90% time-histories (APD ₉₀).

B. prick examine Billy (Zacopride) to ischemia and again the premature beat number, chamber speed persistent period, the chamber that cause of perfusion persistent period, the chamber incidence rate of quivering of quivering all have remarkable inhibitory action, be dose dependent, 50nmol/kg examines the suitableeest administration concentration of Billy (Zacopride) for pricking; It is similar to the anti-arrhythmic lignocaine (lidocaine) of generally acknowledging to suppress ARR effect.

The present invention compared with prior art has the following advantages and effect:

1. pricking and examining Billy (Zacopride) is the selectivity cardiac muscle I that finds first at present _K1Channel agonist, and be used for the research of antiarrhythmic medicine.

2. prick and examine the medicine of Billy (Zacopride) as treatment ischemia-reperfusion arrhythmia, its effect is similar to lignocaine, and effect is stable, and the rhythm of the heart is not had obvious influence, and safety range is big.Bring out in the ARR experiment at ischemia-reperfusion, lignocaine can make the rhythm of the heart slow down, and bundle is examined Billy (Zacopride) does not then have obviously influence; Lignocaine increases by 1/3 o'clock its antiarrhythmic effect at dosage and obviously weakens, and bundle is examined Billy (Zacopride) and this phenomenon then just occurs when 100 times of valid density.

Description of drawings

Fig. 1. prick and examine Billy (Zacopride) rat ventricular myocytes I _K1The influence of electric current.

Among Fig. 1: a: normal control b: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol/L d: prick and examine Billy 30 μ mol/L

Fig. 2. rat ventricular myocytes I _K1The I-V curve of electric current and bundle are examined Billy (Zacopride) to rat ventricular myocytes film I _K1The effect of electric current

Fig. 3. prick and examine Billy (Zacopride) rat ventricular myocytes I _Ca-LThe influence of electric current

Among Fig. 3: a: contrast; B: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol b: prick and examine Billy 30 μ mol/L;

Fig. 4. the I-V curve of rat ventricular myocytes L type calcium channel and bundle are examined Billy (Zacopride) to rat ventricular myocytes I _Ca-LThe influence of electric current

Fig. 5. prick and examine Billy (Zacopride) rat ventricular myocytes I _NaThe influence of electric current.

Among Fig. 5: a: normal control b: prick and examine Billy 30 μ mol/L

Fig. 6. rat ventricular myocytes I _NaThe I-V curve of electric current and bundle are examined the influence of Billy (Zacopride) to the rat ventricular myocytes sodium current

Fig. 7. prick and examine Billy (Zacopride) rat ventricular myocytes I _ToThe influence of electric current.

Among Fig. 7: a: normal control b: prick and examine Billy 30 μ mol/L

Fig. 8. rat ventricular myocytes I _ToThe I-V curve of electric current and bundle are examined Billy (Zacopride) to rat ventricular myocytes film I _ToThe influence of electric current

Fig. 9. prick and examine Billy (Zacopride) rat ventricular myocytes I _Na/CaThe influence of electric current

Among Fig. 9: a: normal control b: prick and examine Billy 30 μ mol/L c:5mmol/LNiCl ₂

Figure 10. prick and examine Billy (Zacopride) the myocyte I of guinea-pig ventricular _KThe influence of electric current

Among Figure 10: a: normal control b: prick and examine Billy 10umol/L

Figure 11. the myocyte I of guinea-pig ventricular _KThe I-V curve of electric current and bundle are examined Billy (Zacopride) to the muscle cell membrane I of guinea-pig ventricular _KThe influence of electric current

Figure 12. prick and examine the influence of Billy (Zacopride) the rat ventricular myocytes action potential.

Among Figure 12: a: normal control b: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol/L d: prick and examine Billy 30 μ mol/L e: prick and examine Billy 40 μ mol/L

Figure 13. variable concentrations is pricked and is examined the premature beat number of Billy (Zacopride) in 15 minute ischemic stage

Figure 14. variable concentrations is pricked and to be examined Billy (Zacopride) group in the quiver comparison of persistent period of inner room speed, chamber of 15 minute ischemic stage.

Figure 15. the electrocardiogram when rat normally reaches ischemic arrhythmia.

Among Figure 15: preceding 2 minutes vena femoralis injections of A ischemic arrhythmia electrocardiogram (not administration) B ischemia are pricked and are examined Billy 50nmol/kg, can effectively prevent preceding 5 minutes vena femoralis injection lignocaine 7.5mg/kg of ischemic arrhythmia C ischemia, preceding 5 minutes vena femoralis injection lignocaine 10mg/kg of ischemic arrhythmia D ischemia can be effectively prevented, ARR generation can be promoted.A: electrocardiogram b before the ischemia: 1 minute c after the left anterior descending coronary artery ligation: premature ventricular beat d: chamber speed e: f quivers in the chamber: the preceding vena femoralis injection lignocaine 10mg/kg of ischemia is decreased heart rate g obviously: lignocaine 10mg/kg antiarrhythmic effect obviously weakens, and quivers and premature beat in the chamber of appearance.

Figure 16. variable concentrations is pricked and to be examined quiver in 15 minutes and reperfusion phase inner room speed, the chamber comparison of persistent period of Billy (Zacopride)

The specific embodiment

Examine Billy (Zacopride) and carry out about to I with pricking below _K1The experimentation of the agonism of passage reaches the discussion to antiarrhythmic effect, and its new purposes at pharmaceutical field is described.

Embodiment 1: bundle is examined Billy, and (Zacopride is to the influence of ventricular muscle cell ion channel current

1 materials and methods:

1.1 laboratory animal:

The healthy SD rat, male, body weight 220-260g is available from Henan Zhengzhou University Experimental Animal Center.

1.2 medicine and reagent:

Zacopride hydrochloride is available from TOCRIS.Collagenase P is available from German Bochringer Mannhein company, taurine (taurine), triethylammonium tetrakis (tetraethylammonium chloride, TEA), 4-aminopyridine (4-aminopyridine, 4-AP), nicardipine (nicardipine), adenosine disodium triphosphate (adenosine 5 '-triphosphate disodium salt), adenosine triphosphate di-potassium (adenosine 5 '-triphosphate dipotassium salt), adenosine triphosphate magnesium salt (adenosine 5 '-triphosphatemagnesium salt), DL-aspartic acid potassium salt (DL-aspartic acid potassium), L-glutamic acid (L-glutamic acid), Ethylene Glycol-bis (beta-aminoethyl-ether)-N, N, N ', N '-Tetra Acetate (EGTA), creatine phosphate sodium salt (creatinine phosphate sodium salt) is available from U.S. Sigma company; HEPES (N-2-hudroxyethylpiperazine-N '-2-ethane-sulfonic acide) available from U.S. sigma company, all the other are domestic production analytical pure product.

1.3 key instrument:

Axopatch 200B patch clamp amplifier (Axon Instrument, USA), Digidate 1322A analog-digital converter (AxonInstrument, USA) and Pclamp8.0 (Axon Instrumen, USA), microelectrode draws instrument (Narishige, PP-83, CO.Japan)

1.4 reagent preparation:

1.4.1 solution preparation:

1. tyrode's solution (mmol/L): NaCl 140, and KCl 5.4, NaH ₂PO ₄0.33 HEPES 5.0, glucose 10, MgCl ₂1.0, CaCl ₂1.8, regulate PH to 7.38 with NaOH.

2. enzyme liquid (mmol/L): NaCl 125, and KCl 5.4, CaCl ₂75, MgCl ₂1.0, NaH ₂PO ₄0.33,4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) 10, glucose 10, and taurine 20, CollagenaseP (Boehringe Mannheim, Germary) collagenase P 0.1-0.3mg/ml (3.5-5mg).

3.KB liquid (mmol/L): KOH 85, L-glutamic acid 50, and KCl 30, and taurine 20, KH ₂PO ₄30, MgCl ₂1.0 HEPES 10, glucose 10, and EGTA0.5 regulates PH to 7.4 with KOH.

4. prick and examine Billy (Zacopride) stock solution: with deionized water dissolving zacopride concentration is 1mmol/L.Dilute on demand during use.

1.4.2 the preparation of liquid in the electrode:

1.L-type calcium current (I _Ca-L) electrode in liquid composition (mmol/L): KCl 150, and HEPES 5, and EGTA 5.0, ATP-K ₂3.0, MgCl ₂1.0 4-AP 5.0, ATP-Mg 1.0, regulate PH to 7.3 with KOH.The extracellular perfusate is a tyrode's solution.

2. liquid composition (mmol/L) in the electrode of instantaneous export-oriented potassium current (Ito): KCl 150, and HEPES 5.0, and EGTA 5.0, ATP-K ₂3.0, MgCl ₂1.0, regulate PH to 7.3 with KOH.The extracellular perfusate is: add CdCl in the tyrode's solution ₂0.1mmol/L, BaCl ₂0.2mml/L, block I respectively _Ca-LAnd inward rectifyimg potassium channel.

3. inward rectification potassium current (I _K1) interior liquid composition of electrode and I _Ca-LIdentical.Extracellular fluid adds CdCl in tyrode's solution ₂0.5mmol/L blocking-up I _Ca-LElectric current.

4. sodium current (I _Na) electrode in liquid (mmol/L): NaCl 5.0, and CsCl 20, and KCl 110, MgCl ₂1.0, HEPES5.0, EGTA 5.0, and Mg-ATP 5.0, regulate PH to 7.2 with CsOH.Extracellular perfusate composition (mmol/L): NaCl 50, CsCl90, MgCl ₂1, glucose 10, CaCl ₂0.5, CoCl ₂0.5 HEPES 5.0, regulate PH to 7.4 with CsOH

5. sodium calcium exchanging electric current (I _Na/Ca) electrode in liquid composition (mmol/L): Aspartic acid potassium 42, MgCl ₂13, EGTA42, CaCl ₂29, ATP-K ₂10, phosphagen sodium salt 5.0, HEPES 5.0, and 4-AP 20, regulate PH to 7.3 with CsOH.Extracellular fluid composition (mmol/L): NaCl 140, CaCl ₂1.8, MgCl ₂2.0 HEPES 5.0, glucose 10 is regulated PH to 7.4 with CsOH, adds nicardipine 1 μ mol/L, ouabain 20 μ mol/L, BaCl ₂1.0mmol/L CsCl 2mmol/L blocks L type calcium channel, sodiumpotassium pump and potassium channel respectively.

2 experimental techniques:

2.1 the separation method of single ventricular muscle cell:

Select healthy adult SD rat 220-260g for use, male, preceding 15 minutes abdominal vein injecting heparins (1000U/kg) of art, pentobarbital sodium (65mg/kg, ip) anesthesia rear neck artery blood-letting, open breast rapidly and take out heart, place 4 ℃ to prune, then heart is suspended on the Langendorff perfusion device through the aorta perfusion that drives in the wrong direction with the saturated no calcium tyrode's solution of 100% oxygen.Use no calcium Zinciodati Comp solution perfusion 8～10 minutes, reuse enzyme liquid circulation perfusion 15～20 minutes earlier.Keep 37 ℃ of constant temperature, perfusion pressure 70cmH in the perfusing course ₂O,, and continue to pass to 100% oxygen.Treat after the ventricular muscles tissue becomes big, deliquescing it to be shredded, place KB liquid blow and beat gently and obtain dispersive ventricular muscle cell, be stored in the KB liquid behind the strainer filtering in 150 μ m apertures, the room temperature placement experimentized in 5～6 hours.

2.2 full cell patch pincers recording method:

The cell that to deposit in earlier before the experiment in the high potassium KB liquid is progressively answered calcium.Get several cell suspension then and add cell pools (about 1ml), lie on the inverted microscope that is connected with little manipulation instrument and leave standstill 10min, treat cell abundant adherent after, use Zinciodati Comp solution perfusion, the about 1～2ml/min of speed.The glass for electrode capillary tube draws instrument through microelectrode, and (Narishige, PP-83 CO.Japan) draw in two steps and form, and charge in the electrode behind the liquid the about 2～5M Ω of resistance.Choose band clearly myocardial cell experimentize.After forming the high impedance sealing-in, use the negative pressure rupture of membranes, carry out full cell record.Ion current signal is through Axopatch 200B patch clamp amplifier (AxonInstrument, USA), Digidate 1322A analog-digital converter (Axon Instrument, USA) and Pclamp8.0 (AxonInstrumen, USA) gather, store and analyze, and be kept in the computer hard disc, all experiments are all carried out under 25 ℃ of room temperatures.

The size of applied current density (being the membrane current of unit membrane electric capacity) expression membrane current.Carry out full cell plasma stream record under the voltage clamp mode, label action potentials of cells under the current clamp mode.

2.3 observation index:

Observe to prick and examine Billy (Zacopride) rat ventricular myocytes L-type Ca ²⁺Electric current (I _Ca-L), I _Na, instantaneous export-oriented potassium current (I _To) and inward rectification potassium current (I _K1) and ventricular muscle cell I _Na/CaInfluence; Observe to prick and examine Billy (Zacopride) the myocyte I of guinea-pig ventricular _KThe influence of electric current; Observe to prick and examine Billy (Zacopride) rat ventricular myocytes transmembrane potential, action potential amplitude (Action potential Amplitude, APA) action potential multipole 50% time-histories (Action potential duration at50% repolarization, APD ₅₀) and action potential multipole 90% time-histories (Action potential duration at 90%repolarization, APD ₉₀) influence.

2.4 date processing:

(Mean ± SD) expression adopts the SPSS11.0 statistical software to carry out one factor analysis of variance to data, and P＜0.05 is for having significant difference with mean ± standard deviation.

3 experimental results

3.1 prick and examine Billy (Zacopride) to rat ventricular myocytes inward rectification potassium current (I _K1) influence

Keep current potential-40mv, give hyperpolarization, continue the phase step type pulse activation I of 470ms to-100mv _K1, in extracellular fluid, add CdCl ₂0.5mmol/L with blocking-up I _Ca-LElectric current.

Bundle is examined Billy (Zacopride) and is concentration dependent enhancing I _K1, 10 μ mol/L bundle is examined Billy (Zacopride) can make I _K1Electric current density increases by 29.45% (P＜0.01).30 μ mol/L bundle is examined Billy (Zacopride) can make I _K1Electric current density increases by 38.53% (P＜0.01).See Table 1, Fig. 1 (among Fig. 1: a: normal control b: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol/Ld: prick and examine Billy 30 μ mol/L), Fig. 2.

Table 1. is pricked and is examined Billy (Zacopride) to rat ventricular myocytes I _K1The influence of electric current.

μmol/L	Cell example number	Electric current density (PA/PF)	Rate of change (%)
				0.0 1.0 3.0 10.0 30.0	6 6 6 6 6	8.69±0.42 8.92±0.91 9.17±1.06 11.25±0.63 ** 12.04±3.66 **	0 +2.60 +5.90 +29.45 +38.53

Compare with matched group ^*P＜0.05 ^*P＜0.01

Examine the influence of Billy (Zacopride) 3.2 prick to each ion channel of rat ventricular myocytes

3.2.1 prick and examine Billy (Zacopride) to rat ventricular myocytes L-type Ca ²⁺Electric current (I _Ca-L) influence

Measure L-type calcium channel electric current (L-type calcium current, I _Ca-L) time, keep current potential-40mv, depolarization is arrived+10mv, and the step pulse that continues 500ms activates I _Ca-Lis for confirming that the electric current that records is I _Ca-L, in extracellular fluid, add 5 μ mol/L nicardipines, can block this electric current fully, thereby support that the electric current that is write down is I _Ca-LI is treated in rupture of membranes back balance 5 minutes _Ca-LStable back adds medicine and experimentizes..

1--30 μ mol/L scope Nei Zhakao Billy (Zacopride) is to I _Ca-LDo not have obviously effect, electric current density is compared no difference of science of statistics, P＞0.05 before and after the medication.See Table 2, Fig. 3 (a: normal control b: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol/Ld: prick and examine Billy 30 μ mol/L), Fig. 4.

Table 2. is pricked and is examined Billy (Zacopride) to rat ventricular myocytes I _Ca-LThe influence of electric current.

μmol/L	Cell example number	Electric current density (PA/PF)	Rate of change (%)
				0 1.0 3.0 10.0 30.0	5 5 5 5 5	11.13±1.13 11.16±1.24 10.91±1.55 11.87±1.36 12.06±1.41	+0.26 -1.98 +1.07 +8.35

3.2.2 prick and examine Billy (Zacopride) to rat ventricular myocytes I _NaInfluence

When surveying sodium current, keep voltage-80mV, depolarization is to 0mV, and the step pulse that continues 50ms activates I _Na

1--30 μ mol/L pricks and examines Billy (Zacopride) to I _NaDo not make significant difference.Electric current density is compared no difference of science of statistics, P＞0.05 before and after the medication.See Table 3, Fig. 5 (among Fig. 5: a: normal control b: prick and examine Billy 30 μ mol/L), Fig. 6.

Table 3. is pricked and is examined Billy (Zacopride) to rat ventricular myocytes I _NaThe influence of electric current

μmol/L	Cell example number	Electric current density (PA/PF)	Rate of change (%)
				0.0 1.0 3.0 10.0 30.0	6 6 6 6 6	38.16±1.46 36.78±1.33 39.21±1.18 37.64±1.39 37.92±1.34	-3.62 +2.75 -1.36 -0.63

3.2.3 prick and examine Billy (Zacopride) to the instantaneous export-oriented potassium current (I of rat ventricular myocytes _To) influence

Measure I _ToThe time, keep voltage-40mV, depolarization is to+50mV, and the step pulse that continues 60ms activates I _To, in extracellular fluid, add CdCl ₂0.5mmol/L, BaCl ₂0.5mmol/L, block I respectively _Ca-LAnd inward rectifyimg potassium channel electric current (I _K1).

Bundle is examined Billy (Zacopride) 1--30 μ mol/L to I _ToThere is not obviously influence.Electric current density is compared no difference of science of statistics, P＞0.05 before and after the medication.See Table 4, Fig. 7 (among Fig. 7: a: normal control b: prick and examine Billy 30 μ mol/L), Fig. 8.

Table 4. is pricked and is examined Billy (Zacopride) to rat ventricular myocytes I _ToThe influence of electric current.

μmol/L	Cell example number	Electric current density (PA/PF)	Rate of change (%)
				0.0 1.0 3.0 10.0 30.0	6 6 6 6 6	24.39±1.23 23.72±1.34 24.41±1.25 23.64±1.12 23.21±1.16	-1.33 +0.31 -3.08 -4.84

3.2.4 prick and examine Billy (Zacopride) to rat ventricular myocytes I _Na/CaInfluence

I _Na/CaMeasure, utilization sawtooth voltage program record current-voltage relationship is kept voltage and is-40mV, and ramp voltage is reduced to-120mV by+60mV, and decrease speed 90mV/s records current-voltage relation, the NiCl of 5mmol/L ₂Make forward and reverse I _Na/CaAll reduce.Utilize NiCl ₂Difference between current before and after the blocking-up obtains Ni ²⁺Responsive electric current is I _Na/Ca

Bundle is examined Billy (Zacopride) to I _Na/CaDo not have obviously influence, compare no difference of science of statistics, P＞0.05 before and after the medication.See Table 5, Fig. 9 is (among Fig. 9: a: normal control b: prick and examine Billy 30 μ mol/L c:5mmol/LNiCl ₂).

Table 5. is pricked and is examined Billy (Zacopride) to rat ventricular myocytes I _Na/CaThe influence of electric current.

μmol/L	Cell example number	Electric current density (PA/PF)
				+70mv	-70mv
		0.0 1.0 3.0 10.0 30.0	6 6 6 6 6			0.86±0.06 0.88±0.07 0.91±0.11 0.88±0.09 0.91±0.03	0.48±0.05 0.47±0.06 0.49±0.03 0.48±0.04 0.47±0.03

3.3 prick and examine Billy (Zacopride) to the myocyte I of guinea-pig ventricular _KInfluence

Because rat ventricular myocytes I _KThe passage distribution density is low, examines Billy (Zacopride) to ventricular muscle cell I so adopt Cavia porcellus to observe bundle in addition _KInfluence, the myocyte of guinea-pig ventricular separation method, rheometer recording method are similar to rat experiment.

The result shows, pricks and examines Billy (Zacopride) to I _KThere is not obviously influence.I before and after the medication _KDensity is compared P＞0.05.See Table 6, Figure 10 (among Figure 10: a: normal control b: prick and examine Billy 10 μ mol/L), Figure 11.

Table 6. is pricked and is examined Billy (Zacopride) to the myocyte I of guinea-pig ventricular _KThe influence of electric current.

μmol/L	Cell example number	Electric current density (PA/PF)	Rate of change (%)
				0.0 1.0 3.0 10.0 30.0	6 6 6 6 6	13.20±1.89 12.78±2.32 12.84±2.16 12.67±1.99 13.01±2.05	-3.19 -3.72 -4.02 -1.44

Examine the influence of Billy (Zacopride) 3.4 prick to the rat ventricular myocytes transmembrane potential

Adopt full cell patch pincers record, with current clamp molding formula record resting potential (Resting potential, RP) and action potential (Action potential, AP), AP is by 3ms, 0.5Hz, the inward electric current of 700pA-1000pA excites to observe bundle respectively and examines Billy (Zacopride) to transmembrane potential action potential amplitude (Action potential Amplitude, APA) action potential multipole 50% time-histories (Action potential duration at 50%repolarization, APD ₅₀) and action potential multipole 90% time-histories (Actionpotential duration at 90%repolarization, APD ₉₀) influence

Bundle is examined Billy (Zacopride) influence of resting potential, action potential amplitude and action potential duration, APD is seen Table 7, Figure 12 (among Figure 12: a: normal control b: prick and examine Billy 3 μ mol/L c: prick and examine Billy 10 μ mol/L d: prick and examine Billy 30 μ mol/L e: prick and examine Billy 40 μ mol/L).

3～40 μ mol/L scope Nei Zhakao Billys (Zacopride) but concentration dependent strengthens resting potential, increase action potential amplitude, and current potential multipole under reach 50% time-histories (APD ₅₀) and action potential multipole 90% time-histories (APD ₉₀).

Table 7 is pricked and is examined the influence of Billy (Zacopride) to rat ventricular myocytes resting potential, action potential amplitude and action potential duration, APD

Concentration (μ mol/L)	Resting membrane electric potential (millivolt)	APA (millivolt)		APD 50(millisecond)		APD 90(millisecond)
								Mean ± standard deviation	Rate of change %	Mean ± standard deviation	Rate of change %	Mean ± standard deviation	Rate of change %
		0 3 10 30 40	-77.8±1.37 -82.4±1.19 *-87.2±2.47 **-90.3±1.72 **-90.7±2.93 **	121.77±3.83 122.98±2.90 130.62±5.94 *141.62±3.57 **143.46±5.79 **	+0.01 +0.16 +0.18 +0.07	9.79±1.24 9.29±3.88 7.82±2.51 * 6.78±1.69 * 6.52±2.38 *	-0.05 -0.20 -0.31 -0.33							18.48±4.70 18.02±2.40 17.61±3.97 * 15.50±5.43 * 5.06±5.43 *	-0.02 -0.05 -0.16 -0.19

Compare with matched group ^*P＜0.05; ^*P＜0.01

Embodiment 2: prick and to examine Billy (Zacopride) Chinese People's Anti-Japanese Military and Political College Mus ischemic, pour into the research of arrhythmia effect again

1 materials and methods

1.1 laboratory animal:

The healthy SD rat, male, body weight 220-260g is available from Henan Zhengzhou University Experimental Animal Center.

1.2 medicine and reagent:

Zacopride hydrochloride is available from TOCRIS.Hydrochloric acid Lidocaine injection is available from Shanghai Xudong Hipu Medicine Co., Ltd.

1.3 key instrument:

Rm6240 multi-path physiology signal acquiring processing system (Chengdu Instruement Factory); HX-200 animal respirator (Chengdu TME Technology Co., Ltd.)

1.4 reagent preparation:

1.4.1 solution preparation:

1. prick and examine Billy (Zacopride) stock solution: with deionized water dissolving zacopride concentration is 1mmol/L.Dilute on demand during use..

2. lignocaine stock solution (0.1g/5ml): take by 7.5mg/kg during use

2 experimental techniques:

2.1 ischemic arrhythmia model preparation method:

2.1.1 experiment grouping:

48 of healthy SD rats are divided into 6 groups at random, and promptly normal saline matched group, bundle are examined Billy (Zacopride) 5nmol/kg group, 15nmol/kg group, 50nmol/kg group and lignocaine (7.5mg/kg) positive controls.

2.1.2 experimental technique:

With rat with pentobarbital sodium (65mg/kg, ip) after the anesthesia, dorsal position is fixed on the operating-table, cervical region median incision then, tracheal intubation connects artificial respirator (DH-1 type); After the common carotid artery intubate, Rm6240 multi-path physiology signal acquiring processing system (Chengdu Instruement Factory) continuous record II lead electrocardiogram and blood pressure.Lock center line the 4th, 5 intercostal otch leave breast in the breastbone left side, the 2mm place after left anterior descending coronary artery (LAD) is sent, and in left auricle lower edge inserting needle, the pulmonary conus place goes out pin with the medical eyeless suture needle of 6-0, and line is penetrated a short PE50 pipe.After stablizing 5min, the femoral vein administration.Strain silk thread behind the 2min, cause myocardial ischemia, continue 15min, continuous record electrocardiogram (ECG) arteriotony (BP) during this period with the terminal compressing of plastic tube coronary artery.Because lignocaine has the effect of decreased heart rate, so in ischemia administration in preceding 5 minutes, all the other methods are the same.

2.1.3 laboratory observation index:

The time that arrhythmia occurs the earliest behind the ischemia (incubation period); The arrhythmia type; PVB: occur the VEA below 5 continuously; VT: occur the ventricular premature contraction more than 5 continuously; VF: quiver in the chamber, disappears for QRS involves the T ripple, for they a series of sizes, variform irregular fluctuation.

PVB number in the record ischemia 15min, VT, VF persistent period (for adding up of VT, VF persistent period occur at every turn).

2.2 pour into arrhythmia model preparation method again

2.2.1 experiment grouping:

48 of healthy SD rats are divided into 6 groups at random, and promptly normal saline matched group, bundle are examined Billy (Zacopride) 1.5nmol/kg group, 5nmol/kg group, 15nmol/kg group and lignocaine (7.5mg/kg) positive controls.

2.2.2 experimental technique:

With rat with pentobarbital sodium (65mg/kg, ip) after the anesthesia, dorsal position is fixed on the operating-table, cervical region median incision then, tracheal intubation connects artificial respirator (DH-1 type); After the common carotid artery intubate, Rm6240 multi-path physiology signal acquiring processing system (Chengdu Instruement Factory) continuous record II lead electrocardiogram and blood pressure.Lock center line the 4th, 5 intercostal otch leave breast in the breastbone left side, the 2mm place after left anterior descending coronary artery (LAD) is sent, and in left auricle lower edge inserting needle, the pulmonary conus place goes out pin with the medical eyeless suture needle of 6-0, and line is penetrated a short PE50 pipe.After stablizing 5min, the femoral vein administration.Strain silk thread behind the 2min, cause myocardial ischemia, continue to unclamp silk thread after 15 minutes, poured into again 15 minutes, during this period continuous record electrocardiogram (ECG) and arteriotony (BP) with the terminal compressing of plastic tube coronary artery.Gave normal saline respectively by femoral vein in preceding 2 minutes in pouring into again, prick and examine each dosage of Billy (Zacopride).Because lignocaine has the effect of decreased heart rate, so pouring into administration in preceding 5 minutes again, all the other methods are the same.

2.2.3 observation index:

Pour into back arrhythmia type: PVB: occur the VEA below 5 continuously again; VT: occur the ventricular premature contraction more than 5 continuously; VF: quiver in the chamber, disappears for QRS involves the T ripple, for they a series of sizes, variform irregular fluctuation.

Record pours into the PVB number in the 15min again, VT, VF persistent period (for adding up of VT, VF persistent period occur at every turn).

2.4 date processing:

(Mean ± SD) expression adopts the SPSS11.0 statistical software to carry out one factor analysis of variance to data, and P＜0.05 is for having significant difference with mean ± standard deviation.

3 experimental results

3.1 ischemic arrhythmia

Premature beat, chamber speed all appear in 8 animals of normal saline matched group behind the ischemia, have 6 generation chamber to quiver.Bundle examine Billy (Zacopride) to ischemia and again premature beat number, the chamber speed persistent period, the chamber that cause of perfusion persistent period, the chamber incidence rate of quivering of quivering all have remarkable inhibitory action, be dose dependent.Originally studies show that lignocaine 7.5mg/kg is the suitableeest administration concentration, when dosage strengthens (10mg/kg), find its obviously decreasing heart rate, antiarrhythmic effect obviously weakens.The result shows that the 50nmol/kg bundle is examined Billy and compared its antiarrhythmic effect no difference of science of statistics with the 7.5mg/kg lignocaine.

The results are shown in Table 8, (among Figure 15: preceding 2 minutes vena femoralis injections of A ischemic arrhythmia electrocardiogram (not administration) B ischemia are pricked and are examined Billy 50nmol/kg for Figure 13, Figure 14, Figure 15, can effectively prevent preceding 5 minutes vena femoralis injection lignocaine 7.5mg/kg of ischemic arrhythmia C ischemia, preceding 5 minutes vena femoralis injection lignocaine 10mg/kg of ischemic arrhythmia D ischemia can be effectively prevented, ARR generation can be promoted.

A: electrocardiogram b before the ischemia: 1 minute c after the left anterior descending coronary artery ligation: premature ventricular beat d: chamber speed e: f quivers in the chamber: the preceding vena femoralis injection lignocaine 10mg/kg of ischemia is decreased heart rate obviously) g: lignocaine 10mg/kg antiarrhythmic effect obviously weakens, and quivers and premature beat in the chamber of appearance.)

Table 8. is pricked and is examined the influence of Billy (zacopride) to the anesthetized rat ischemic arrhythmia

Medicine	Number of elements	Arrhythmia incubation period (sec)	The premature beat number	The chamber speed persistent period (sec)	(sec) quivers the persistent period in the chamber	The chamber incidence rate (%) of quivering
							Bundle is examined Billy 0nmol/kg 5nmol/kg 15nmol/kg 50nmol/kg lignocaine 7.5 mg/kg	8 8 8 8 8	344.13±59.79 394.25±51.48 406.13±77.88 475.63±99.06 *△ 518.75±78.63 *	149±28 142±26 50±15 *45±21 *△41±13 *	51.18±16.56 40.44±14.79 8.69±4.24 * 2.09±1.91 *△ 1.21±1.57 *	6.90±7.05 0.69±1.94 * 0 * 0 *△ 0.23±0.64 *	75 12.5 0 0 12.5

^*Compare P＜0.01 with matched group, ^△Compare P＞0.05 with the lignocaine group

3.2 pour into arrhythmia again

Premature beat, chamber speed all appear in 8 animals of normal saline matched group behind the ischemia-reperfusion, have 7 generation chamber to quiver.Bundle is examined premature beat number, chamber speed persistent period, chamber that Billy occurs after to ischemia-reperfusion persistent period and the chamber incidence rate of quivering of quivering all the dose-dependent inhibition effect.The 5nmol/kg group is examined the suitableeest dosage of Billy for pricking, and the 5nmol/kg bundle is examined Billy and compared with the 7.5mg/kg lignocaine, and it suppresses to pour into arrhythmia effect no difference of science of statistics again.The results are shown in Table 9, Figure 16.

Table 9. is pricked and is examined Billy (zacopride) pours into arrhythmia again to anesthetized rat influence

Medicine	Number of animals (only)	The premature beat number	The chamber speed persistent period (sec)	(sec) quivers the persistent period in the chamber	The chamber incidence rate (%) of quivering
						Bundle is examined Billy 0nmol/kg 1.5nmol/kg 5nmol/kg 15nmol/kg lignocaine 7.5mg/kg	8 8 8 8 8	23±14 23±18 35±33 △ 26±15 22±11	13.35±4.98 6.97±3.68 5.10±2.62 *△ 6.19±2.89 * 2.71±2.75 *	11.72±4.91 6.50±2.92 1.25±3.54 *△ 3.13±1.94 * 0.95±1.69 *	87.5 75 12.5 25 12.5

^*Compare P＜0.05 with matched group, ^△Compare P＞0.05 with the lignocaine group

Positive controls lignocaine dosage is 7.5mg/kg in this experiment, when dosage strengthens (10mg/kg), finds its obviously decreasing heart rate, and antiarrhythmic effect obviously weakens, and the drug safety scope is little.The results are shown in Figure 15 (D:f, g).

Claims (2)

1. prick and examine the application of Billy as preparation treatment antiarrhythmic medicament.

2. bundle according to claim 1 is examined the application of Billy as preparation treatment antiarrhythmic medicament, it is characterized in that it is capsule, microcapsule, liposome, granule, injection, tablet and oral liquid that described bundle is examined Billy's preparation.

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