CN104055759A - Medicine composition and application thereof - Google Patents
Medicine composition and application thereof Download PDFInfo
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- CN104055759A CN104055759A CN201410267131.6A CN201410267131A CN104055759A CN 104055759 A CN104055759 A CN 104055759A CN 201410267131 A CN201410267131 A CN 201410267131A CN 104055759 A CN104055759 A CN 104055759A
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to the technical field of medicines, and in particular relates to a medicine composition and an application thereof. The medicine composition comprises atomoxetine hydrochloride and taurine, wherein the synergy can be generated via the combination of the atomoxetine hydrochloride and the taurine. The experiment finds that compared with the single application of the atomoxetine hydrochloride, according to the medicine composition prepared by the combination of the atomoxetine hydrochloride and the taurine, the SOD (Superoxide Dismutase) activity and the GSH (Glutathione) content of the brain tissue of an ADHD (Attention Deficit Hyperactivity Disorder) model rat at a juvenile stage can be obviously increased while the MDA (Methane Dicarboxylic Aldehyde) content (P is less than 0.01) in the brain tissue of the ADHD model rat is decreased, thereby performing an antioxidation function, improving the expressions of DBH (Dopamine Beta-Hydroxylase) and TH (Tyrosine Hydroxylase) in the brain of the ADHD model rat at the juvenile stage and improving the expression (P is less than 0.01) of Fos protein in the prefrontal cortex of the ADHD model rat. Compared with the single application of the medicine, the medicine composition provided by the invention has obvious effects. Thus, the medicine composition can serve as a candidate medicine for treating pediatric hyperactivity.
Description
Technical field
The present invention relates to technical field of pharmaceuticals, relate in particular to a kind of pharmaceutical composition and application thereof.
Background technology
Hyperkinetic syndrome claims again hyperkinetic syndrome (ADHD) or minimal brain dysfunciton syndrome, is that a kind of common child is abnormal diseases, and major effect is to cause children's's dystropy.The main clinical manifestation of hyperkinetic syndrome is: attention deficit disorder, hyperkinesia, perceptual disturbance, dysthymic disorder, maladjustment, learning difficulty etc.Its pathogenic factor probably has following several: cranial nerve dysplasia, brain neurotransmitter lazy weight, cerebral tissue organic lesion, inherited genetic factors, other factors etc.Be mainly in tecnology length of time, the age bracket that prevalence is the highest is 6-12 year.
At present a lot of research shows that children's attention defect hyperkinetic syndrome (ADHD) is mainly due to the slight cerebral function defect due to dopamine in central nervous system (DA) and norepinephrine (NA) dysbolismus.Research in recent years shows: oxidative stress is one of child ADHA pathogenesis, and SOD (superoxide dismutase), GSH (glutathion), MDA (malonaldehyde), BDNF (Brain Derived Neurotrophic Factor), AChE (acetylcholinesterase), DBH (dopamine-β-hydroxylase), TH (TYR hydroxylase), Fos (oligofructose) are the biochemical indicators relevant to oxidative stress.Improve DBH and TH and Fos protein expression noradrenergic neuron and dopaminergic neuron are had to protective effect.
And the medicine of hyperkinetic syndrome can be divided into several classes such as non-central stimulant, central nervous excitation agent, resist melancholy agent, psychosis and Anti-epileptics substantially, concrete clinical application has tomoxetine hydrochloride, methylphenidate, dextro-amphetamine, azoxodone, caffeine, imipramine etc.
Tomoxetine hydrochloride (trade name is selected and thought to reach), it is strong presynaptic noradrenaline transporter body inhibitor, can selectivity suppress presynaptic amine pump to the absorbing again of norepinephrine, thereby improve the symptom of ADHD, indirectly promote completing of understanding and concentrating of attention.But tomoxetine hydrochloride is very weak to other monoamine transporters or receptor affinity, can not solve hyperkinetic syndrome children's brain neuroblastoma and grow problem, there is the problem of " curing the symptoms, not the disease ".
Taurine (Taurine) claim again 2-aminoethyl sulfonic acid, is a kind of non-albumen of sulfur-bearing, exists in vivo with free state, do not participate in the biosynthesis of albumen in body.The rich content of taurine in brain, widely distributed, can obviously promote neural growth promoter and cell proliferation, differentiation, and be dose dependent, in cranial nerve cell growth course, plays an important role.In the animal experiment study of taurine and brain development relation, find, taurine can promote the learning and m emaory of rat.Supplement appropriate taurine and not only can improve learning and memory speed, but also can improve the accuracy of learning and memory, and neural defying age is also had to certain effect.
But, at present not about tomoxetine hydrochloride and taurine coupling are used for the treatment of to the research of hyperkinetic syndrome, and study the two coupling, be used for the treatment of hyperkinetic syndrome had to good prospect and using value.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of pharmaceutical composition and application thereof, and this pharmaceutical composition comprises tomoxetine hydrochloride and taurine, and the two coupling can produce synergistic function.By increasing SOD activity and GSH content in cerebral tissue, lower MDA content, raise DBH, TH or Fos expression, thereby reach the object for the treatment of hyperkinetic syndrome.
The invention provides a kind of pharmaceutical composition, comprising: tomoxetine hydrochloride and taurine.
As preferably, in pharmaceutical composition provided by the invention, the mass ratio of tomoxetine hydrochloride and taurine is 1:0.5～50.
Preferably, in pharmaceutical composition provided by the invention, the mass ratio of tomoxetine hydrochloride and taurine is 1:5～45.
Pharmaceutical composition provided by the invention is the application in SOD accelerative activator within preparing brain.
Pharmaceutical composition provided by the invention is the application in MDA inhibitor within preparing brain.
Application in the medicine of pharmaceutical composition provided by the invention GSH content in preparation increases brain.
The present invention proves by experiment; give separately tomoxetine hydrochloride; can increase SOD activity and GSH content in juvenile stage ADHD rat model cerebral tissue, lower MDA content (P<0.05); and employing taurine and tomoxetine hydrochloride coupling; can significantly increase SOD activity and GSH content in juvenile stage ADHD rat model cerebral tissue, lower MDA content (P<0.01); played antioxidative effect
The application of pharmaceutical composition provided by the invention in the medicine of preparation promotion DBH, TH or Fos expression.
The present invention proves by experiment; give separately tomoxetine hydrochloride; can improve DBH and TH expression in juvenile stage ADHD rat model brain; raise Fos protein expression (P<0.05) in Prefrontal Cortex cortex; and employing taurine and tomoxetine hydrochloride coupling; can more significantly improve DBH and TH expression in juvenile stage ADHD rat model brain, raise Fos protein expression (P<0.01) in Prefrontal Cortex cortex.Show that taurine and tomoxetine coupling have protective effect to the noradrenergic neuron of ADHD rat model and dopaminergic neuron.
The application of pharmaceutical composition provided by the invention in the medicine of preparation treatment hyperkinetic syndrome.
Because SOD is active and GSH content, MDA level, BDH, TH or Fos express with hyperkinetic syndrome closely related, at pharmaceutical composition provided by the invention, can promote that SOD is active, improve GSH content, suppress MDA level, promote BDH, TH or Fos to express, therefore, pharmaceutical composition provided by the invention can be used in the medicine of preparation treatment hyperkinetic syndrome.And the present invention confirms by experiment, compares with giving separately tomoxetine hydrochloride, taurine and tomoxetine hydrochloride coupling significantly (P<0.05) reduce the spontaneous hyperkinetic behavior of rat in new environment.
The present invention also provides a kind of medicine for the treatment of hyperkinetic syndrome, comprises pharmaceutical composition provided by the invention and pharmaceutically acceptable adjuvant.
As preferably, the medicine for the treatment of hyperkinetic syndrome provided by the invention is oral formulations.
Preferably, oral formulations is tablet, capsule, pill, granule, decoction, unguentum, distillate medicinal water, oral solutions, drop pill or syrup.
Preferred, oral formulations is tablet.
As preferably, pharmaceutically acceptable adjuvant is excipient, filler and lubricant.
Preferably, excipient is mannitol; Filler is starch; Lubricant is magnesium stearate and micropowder silica gel.
As preferably, in the medicine for the treatment of hyperkinetic syndrome provided by the invention, the mass fraction of tomoxetine hydrochloride is 2%～50%; The mass fraction of described taurine is 2%～50%.
Preferably, the mass fraction of tomoxetine hydrochloride is 16.5%～18.9%; The mass fraction of described taurine is 14.2%～16.5%.
As preferably, in the medicine for the treatment of hyperkinetic syndrome provided by the invention, wherein the mass parts of each component is:
100 parts of tomoxetine hydrochloride;
75 parts～100 parts of taurines;
275 parts～300 parts, mannitol;
75 parts～100 parts of starch;
2.5 parts～3 parts of magnesium stearate;
1.5 parts～2 parts of micropowder silica gels.
As preferably, the dosage of the medicine for the treatment of hyperkinetic syndrome provided by the invention is 10mg/kg every day.
The invention provides a kind of pharmaceutical composition, comprising: tomoxetine hydrochloride and taurine.The two coupling can produce synergistic function.The present invention proves by experiment; with give separately tomoxetine hydrochloride and compare; adopt taurine and tomoxetine hydrochloride coupling; significantly (P<0.01) increases SOD activity and GSH content in juvenile stage ADHD rat model cerebral tissue, also significantly (P<0.01) lowers MDA level; played antioxidative effect; and significantly (P<0.01) improves DBH and TH expression in juvenile stage ADHD rat model brain, and significantly (P<0.01) raises Fos protein expression in Prefrontal Cortex cortex.Show that taurine and tomoxetine coupling have protective effect to the noradrenergic neuron of ADHD rat model and dopaminergic neuron.Therefore, the present invention also provides the medicine of this pharmaceutical composition for the preparation for the treatment of hyperkinetic syndrome.
Accompanying drawing explanation
Fig. 1 shows the DBH SABC testing result of different disposal SHR rat ADHD model; Wherein: Fig. 1-a shows negative control group rat DBH SABC testing result; Fig. 1-b shows blank group rat DBH SABC testing result; Fig. 1-c shows positive controls 1 rat DBH SABC testing result; Fig. 1-d shows positive controls 2 rat DBH SABC testing results; Fig. 1-e shows experimental group 1 rat DBH SABC testing result;
Fig. 2 shows the Fos SABC testing result of different disposal SHR rat ADHD model; Wherein: Fig. 2-a shows negative control group rat Fos SABC testing result; Fig. 2-b shows blank group rat Fos SABC testing result; Fig. 2-c shows positive controls 1 rat Fos SABC testing result; Fig. 2-d shows positive controls 2 rat Fos SABC testing results; Fig. 2-e shows experimental group 1 rat Fos SABC testing result;
Fig. 3 shows different disposal 6-OHDA neonate rat ADHD model TH SABC testing result; Wherein: Fig. 3-a shows negative control group rat TH SABC testing result; Fig. 3-b shows blank group rat TH SABC testing result; Fig. 3-c shows positive controls 1 rat TH SABC testing result; Fig. 3-d shows positive controls 2 rat TH SABC testing results; Fig. 3-e shows experimental group 1 rat TH SABC testing result;
Fig. 4 shows the Fos SABC testing result of different disposal 6-OHDA neonate rat ADHD model: wherein: Fig. 4-a shows negative control group rat Fos SABC testing result; Fig. 4-b shows blank group rat Fos SABC testing result; Fig. 4-c shows positive controls 1 rat Fos SABC testing result; Fig. 4-d shows positive controls 2 rat Fos SABC testing results; Fig. 4-e shows experimental group 1 rat Fos SABC testing result.
The specific embodiment
The invention provides a kind of pharmaceutical composition and application thereof, those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
Below in conjunction with embodiment, further set forth the present invention:
The impact of embodiment 1 pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model
1), experimental design
Get 9 of 54, newborn 21 days SHR rat ADHD models and WKY rats, adaptability was fed after 3 days, selected 52 SHR rats and by body weight, will be divided at random 6 groups, 8～9/group.
Experiment is with the negative contrast of WKY rat;
The SHR rat ADHD model of not administration of take is blank;
Only to give the positive matched group of rat of tomoxetine hydrochloride;
And establish three experimental grouies of different dosing dosage: wherein, experimental group 1 gives the pharmaceutical composition of high dose, the pharmaceutical composition that experimental group 2 gives middle dosage, the pharmaceutical composition that experimental group 3 gives low dosage.Dosage is as shown in table 1:
According to table 1 dosage, every day gastric infusion once, successive administration 2 weeks (PD23～PD37).Administration volume is all 10mL/kg, and negative control group and blank group give equal-volume distilled water.Within every three days, weigh, by body weight, adjust administration volume.
The grouping of table 1 animal and dosage regimen
According to table 1 grouping and dosage regimen, carry out open field test, the Y maze experiment of juvenile stage SHR rat, and detect physiology, the biochemical indicator of SHR rat, analyze the impact on SHR rat ADHD model on pharmaceutical composition provided by the invention.
2), the impact of pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model open field test
(Panlab company, Spain) hyperkinetic behavior of juvenile stage SHR rat is respectively organized in test to adopt Open-field system.Spacious experiment carried out in the room of 30 ℃ of constant temperature, sound insulation and low-light levels.After rat being put into the center of spacious experimental box (120cm * 120cm * 50cm) of uncovered, start timing, photographic head records the motion path of rat automatically, every animal follow-on test 5 minutes.By every animal in Smart3.0 computed in software 5 minutes, enter the number of times of middle section (60cm * 60cm) and at the percentage ratio of the middle section time of staying.Result represents with mean ± standard deviation, the significant difference between SPSS16.0 software one-way ANOVA analysis bank, and result is as shown in table 2.
The impact of table 2 pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model open field test
| Group | Enter the number of times (inferior) of central area | At central area time of staying percentage ratio (%) |
| Negative control | 12.3±1.5 ** | 2.1±0.8 ** |
| Blank | 101.8±3.0 | 18.4±3.1 |
| Positive controls | 66.6±1.8 * | 12.8±2.4 * |
| Experimental group 1 | 23.2±1.5 ** | 5.1±1.2 ** |
| Experimental group 2 | 25.1±1.8 ** | 5.9±1.0 ** |
| Experimental group 3 | 26.3±1.1 ** | 7.4±1.5 ** |
Note: * shows the P<0.05 that compares with blank group, and * * shows the P<0.01 that compares with blank group
As shown in table 2, compare with blank group, use separately tomoxetine hydrochloride, significantly (P<0.05) reduces that rat enters spacious experimental box central area number of times and at central area time of staying percentage ratio.Pharmaceutical composition provided by the invention can be significantly (P<0.01) reduce that rat enters spacious experimental box central area number of times and at central area time of staying percentage ratio.Show that pharmaceutical composition provided by the invention can significantly suppress the hyperkinetic behavior of SHR rat ADHD model, the more independent medication of effect that two medicine couplings produce is more obvious, illustrates that compositions can produce synergistic function.
3), the impact of pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model Y maze experiment
Adopt the ability of learning and memory of the random not rest method test of Y maze experiment fixed number of times rat.MG-2C/3C type Y labyrinth (Zhangjagang City Biomedical Instruments factory) is comprised of controller and the lost case (I, II, three isometric labyrinth arms of III) of Y type.At the bottom of case, by electric grid, laid and formed, stimulus signal lamp is equipped with on every arm top.Controller has the button of regulation output voltage and for selecting four buttons (I, II, III, 0) of Bei He junctional area, labyrinth.Press at random I, II, during III button, the stimulus signal lamp of respective arms is bright, and this arm is cold place of safety, and junctional area and other two arms of energising are non-security district.Press 0 o'clock, junctional area energising, three arms are no power all.When training experiment starts, random selection one arm is place of safety, rat is put into and adapts to 2 minutes, press at random subsequently other two arm buttons, after rat irriate, can escape, rat is directly gone to place of safety and be defined as " correct response ", other situations are " wrong reaction " (as: running to without light district or reverse running).Follow-on test 10 times.After 24 hours, ditto formally test, measure rat correct response number of times, with accuracy, evaluate the ability of learning and memory that rat escapes electricity irritation:
Accuracy=correct response number of times/10 * 100%
Result represents with mean ± standard deviation, the significant difference between SPSS16.0 software one-way ANOVA analysis bank, and result is as shown in table 3:
The impact of table 3 pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model Y maze experiment
| Group | Accuracy (%) |
| Negative control | 93.5±2.2 ** |
| Blank | 36.7±1.9 |
| Positive controls | 52.3±2.5 * |
| Experimental group 1 | 75.9±2.5 ** |
| Experimental group 2 | 76.5±3.1 ** |
| Experimental group 3 | 78.8±2.4 ** |
Note: * shows the P<0.05 that compares with blank group, and * * shows the P<0.01 that compares with blank group
As shown in table 3, compare with blank group, use separately tomoxetine hydrochloride, significantly (P<0.05) improves the accuracy of rat Y maze experiment, pharmaceutical composition provided by the invention, significantly (P<0.01) improves the accuracy of rat Y maze experiment, and the more independent medicine of effect of compositions is more obvious.Show that two medicine couplings produce obvious synergistic function.
4), the impact of pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model physical signs
After last administration 24 hours, weigh.Adopt the noinvasive tail cover method of RBP-1B type rat blood pressure instrument (Chinese-Japanese Friendship Clinic Medical Inst) to measure Conscious Rat blood pressure and heart rate.Rat is put in blood pressure instrument preheating 5 minutes, the pressure arteries and veins cover of blood-pressure measuring appliance is overlapped to rat tails proximal part, transducer and is placed in upper 1/3 place of tail, resonance-amplifier sensitivity makes pulse wave be advisable to 2-3cm, after regular pulse wave occurs, by inflatable ball, making to press arteries and veins cover internal pressure to be elevated to pulse wave disappears completely, the rising 30mmHg that pressurizes again, then slowly exits by balloon valve, from starting to be deflated to manifold pressure, is 0 maintenance 5-6 second.Examine and read pulse wave from the force value without corresponding when just starting to occur first ripple, this is systolic pressure, reads the heart rate value on monitor simultaneously.Result represents with mean ± standard deviation, and the significant difference between SPSS16.0 software one-wayANOVA analysis bank, the results are shown in Table 4.
The impact of table 4 pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model physical signs
As shown in table 4, give pharmaceutical composition provided by the invention and the physical signs of juvenile stage SHR rat ADHD model is not made a significant impact.
5), the impact of pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model biochemical indicator
A) cerebral tissue specimen collection: get each group through the rat of open field test and Y maze experiment, with 3.5% chloral hydrate deep anaesthesia, open thoracic cavity, 4 ℃ of normal saline of cardiac perfusion, get cerebral tissue, left side cerebral tissue is frozen in-80 ℃, detects for biochemical indicator; Right side brain is fixed on formalin, and routine paraffin wax embedding, section detect for immunohistochemistry.
B) biochemical indicator detects: oxidative stress is one of child ADHA pathogenesis, and therefore, the present invention has detected the biochemical indicator relevant to oxidative stress.According to Nanjing, build up test kit description mensuration cerebral tissue MDA and GSH assay and SOD and the AChE enzymatic activity that Bioengineering Research Institute provides; According to Xi Tang bio tech ltd, Shanghai description, measure cerebral tissue BDNF content, and calculate the relative amount that each index is compared with blank group.Statistical result is as shown in table 5:
The impact of table 5 pharmaceutical composition provided by the invention on juvenile stage SHR rat ADHD model biochemical indicator
Note: * shows the P<0.05 that compares with blank group, and * * shows the P<0.01 that compares with blank group
As shown in table 5, compare with blank group, use separately tomoxetine hydrochloride, can significantly increase SOD activity, GSH content, minimizing MDA content (P<0.05), and pharmaceutical composition provided by the invention significantly (P<0.01) improve that SOD in rat cerebral tissue is active, GSH content, reduce MDA content.Therefore explanation, compares with independent use tomoxetine hydrochloride or taurine, and the antioxidation of drug regimen deposits yields provided by the invention is more obvious, and two medicine couplings produce synergism.
6), immunohistochemical experiment is identified the impact of pharmaceutical composition provided by the invention on BDH, TH and Fos in juvenile stage SHR rat ADHD model cerebral cortex
Each group rat brain paraffin section is positioned in 55 ℃ of baking boxs and is baked 2 hours, being placed in dimethylbenzene stock solution (three parts of I, II, III) dewaxes respectively 2 minutes, with gradient concentration ethanol (100%～70%) rehydration from high to low each 1 minute, distilled water cleaned 5 minutes * 3 times again.3% H2O2 (fresh configuration) at room temperature lucifuge is hatched 20 minutes, and in order to deactivating endogenous peroxydase, distilled water cleans 5 minutes * 3 times.Put antigen retrieval liquid mesohigh and repair 3 minutes, after taking-up, be placed to natural cooling.PBS cleans 5 minutes * 3 times, drips 5%BSA confining liquid, is placed in wet 37 ℃ of incubators of box and hatches 1 minute.Suck tissue unnecessary liquid around, drip PBS liquid dilution primary antibodie DBH (1:100) or Fos (1:50) or TH (1:50), hatch after 1 hour dislocation in 4 ℃ of refrigerator overnight for 37 ℃.PBS liquid cleans 5 minutes * 3 times.Drip biotinylation two anti-, in 37 ℃, hatch 1 hour.PBS cleans 5 minutes * 3 times.Blot PBS, drip biotinylation goat anti-rabbit igg two anti-, hatch 1 hour for 37 ℃.PBS cleans 5 minutes * 3 times.Drip DAB developer, examine under a microscope and control chromogenic reaction, use distilled water cessation reaction in good time.Haematoxylin is redyed; Hydrochloride alcohol differentiation, tap water returns indigo plant; Conventional gradient ethanol (75%～100%) dewaters, dimethylbenzene is transparent, neutral gum mounting.Under microscope, blind method is observed ImmunohistochemistryResults Results, according to experimenter's semi-quantitative method in early stage, under high power lens (* 40), count respectively under each visual field, 3, district of large brain striatum brain DBH-positive neuron cell number and Fos-positive cell number (Yu in TH-positive neuron cell number and the visual field, 3, Prefrontal Cortex cortex brain district, et al., 2013).Result represents with mean ± standard deviation, and the significant difference between SPSS16.0 software one-way ANOVA analysis bank, the results are shown in Figure 1～2.Statistical result is as shown in table 6:
The impact of table 6 pharmaceutical composition provided by the invention on BDH, TH and Fos in juvenile stage SHR rat ADHD model cerebral cortex
| Group | DBH positive cell number | TH positive cell number | Fos positive cell number |
| Negative control | 52.3±1.0 ** | 17.9±0.8 ** | 28.6±1.2 ** |
| Blank | 16.7±1.5 | 2.7±0.6 | 7.5±0.9 |
| Positive controls | 31.2±1.8 * | 6.6±1.0 * | 17.6±1.1 * |
| Experimental group 1 | 45.1±1.4 ** | 12.8±0.5 ** | 27.3±1.1 ** |
| Experimental group 2 | 43.2±1.1 ** | 12.5±0.6 ** | 25.5±1.3 ** |
| Experimental group 3 | 48.3±1.0 ** | 13.5±0.8 ** | 26.3±0.9 ** |
Note: * shows the P<0.05 that compares with blank group, and * * shows the P<0.01 that compares with blank group
As shown in table 5; compare with blank group; use separately tomoxetine hydrochloride significantly (P<0.05) rise brain DBH, TH and Fos expression; and significantly (P<0.01) rise brain DBH, TH and Fos expression of pharmaceutical composition provided by the invention; prompting tomoxetine hydrochloride, taurine or taurine and tomoxetine coupling have protective effect to the noradrenergic neuron of ADHD rat and dopaminergic neuron; and medicine and medicine comparison separately, its more remarkable effect are closed in two medicine couplings.Embodiment 2 pharmaceutical composition provided by the invention affects 1 to 6-OHDA neonate rat ADHD model), animal model and grouping administration
Until neonate rat, in the time of 5 days, be divided at random 7 groups, be followed successively by: Normal group, model control group, positive controls, experimental group 1～3.
Except Normal group, all the other each group respectively subcutaneous injection 0.1ml/10g hydrochloric acid desipramine solution (2.5mg/ml), after 40 minutes, modeling rat is in hypothermic anesthesia on ice 5 minutes, an intracisternal injection 20 μ l/ 6-OHDA solution (5mg/ml); Rats in normal control group is after anaesthetizing 5 minutes on ice, and equal-volume solvent is injected in brain pond.
It is as shown in table 7 that each organizes administration situation, every day gastric infusion once, successive administration 21 days (PD5～PD26).Administration volume is all 0.05mL/10g.Within every three days, weigh, by body weight, adjust administration volume.
The grouping of table 7 animal and dosage regimen
2), the impact of pharmaceutical composition provided by the invention on 6-OHDA neonate rat ADHD model body weight
After administration 21 days, weigh.Result represents with mean ± standard deviation, and the significant difference between SPSS16.0 software one-wayANOVA analysis bank, the results are shown in Table 8.
The impact of table 8 pharmaceutical composition provided by the invention on 6-OHDA neonate rat ADHD model body weight
Note: * * shows and the P<0.01 that compares with Normal group
As shown in table 8, the body weight of administration 6-OHDA model group rat after 21 days obviously alleviates (P<0.01); PD26, single gives tomoxetine (1mg/kg) body weight is had no to impact; PD5～PD26 gives respectively the 6-OHDA-rat of 33mg/kg/d, 100mg/kg/d or 300mg/kg/d taurine, and body weight all has the growth of >10%.
3), the impact of pharmaceutical composition provided by the invention on 6-OHDA neonate rat ADHD model hyperkinetic behavior
After tomoxetine administration 1 hour, adopt open field test to evaluate the spontaneous hyperkinetic behavior activeness of rat in new environment.Prologue proof box (100cm * 100cm * 50cm) is uncovered black experimental box, divides 25 grids (20cm * 20cm) be comprised of white line.When experiment starts, rat is put into case, record rat and wear lattice number of times in 5 minutes.Result represents with mean ± standard deviation, the significant difference between SPSS16.0 software one-wayANOVA analysis bank.Result is as shown in table 9.
The impact of table 9 pharmaceutical composition provided by the invention on 6-OHDA neonate rat ADHD model hyperkinetic behavior
| Group | Wear lattice number of times (inferior) |
| Normal control | 48.5±1.3 |
| Model contrast | 100.2±1.9 ## |
| Positive controls | 55.6±1.5 * |
| Experimental group 1 | 33.1±1.1 ** |
| Experimental group 2 | 34.3±1.0 ** |
| Experimental group 3 | 31.8±1.3 ** |
##show the P<0.01 that compares with Normal group,
*show the P<0.05 that compares with model group,
*show the P<0.01 that compares with model group
As shown in table 9, to compare with Normal group, model group rat is worn lattice number of times significantly increases (P<0.01).Compare with model control group, use separately tomoxetine hydrochloride significantly (P<0.05) reduce rat and wear lattice number of times, and pharmaceutical composition provided by the invention significantly (P<0.01) reduction rat wear lattice number of times.Show that pharmaceutical composition provided by the invention can significantly suppress the hyperkinetic behavior of SHR rat ADHD model.
4), the invention provides the impact of pharmaceutical composition on 6-OHDA neonate rat ADHD model biochemical indicator
A) cerebral tissue specimen collection: get the wear long rat of lattice experiments of each group, 3.5% chloral hydrate deep anaesthesia, opens thoracic cavity, 4 ℃ of normal saline of cardiac perfusion, gets cerebral tissue, and left side cerebral tissue is frozen in-80 ℃, detects for biochemical indicator; Right side brain is fixed on formalin, and routine paraffin wax embedding, section detect for immunohistochemistry.
B) biochemical indicator detects: according to Nanjing, build up the test kit description mensuration cerebral tissue MDA content that Bioengineering Research Institute provides; According to Xi Tang bio tech ltd, Shanghai description, measure cerebral tissue BDNF content, result is as shown in table 10.
The impact of table 10 pharmaceutical composition provided by the invention on 6-OHDA neonate rat ADHD model biochemical indicator
##show the P<0.01 that compares with Normal group,
*show the P<0.05 that compares with model group,
*show the P<0.01 that compares with model group
As shown in table 10, compare with model group, use separately tomoxetine hydrochloride can reduce MDA content (P>0.05), rising BDNF content (P<0.05).And pharmaceutical composition provided by the invention significantly reduces MDA content in rat cerebral tissue, rising BDNF content.Result shows, compares with independent use tomoxetine hydrochloride, and pharmaceutical composition provided by the invention can produce more obvious antioxidation, and two medicines share and independent medicine comparison, its more remarkable effect.
5), immunohistochemical experiment identifies that pharmaceutical composition provided by the invention is to 6-OHDA neonate rat ADHD
The impact of BDH, TH and Fos in model cerebral cortex
The brain paraffin section of each group rat is positioned in 55 ℃ of baking boxs and is baked 2 hours, being placed in dimethylbenzene stock solution (three parts of I, II, III) dewaxes respectively 2 minutes, with gradient concentration ethanol (100%～70%) rehydration from high to low each 1 minute, distilled water cleaned 5 minutes * 3 times again.3% H
2o
2(fresh configuration) at room temperature lucifuge hatches 20 minutes, and in order to deactivating endogenous peroxydase, distilled water cleans 5 minutes * 3 times.Put antigen retrieval liquid mesohigh and repair 3 minutes, after taking-up, be placed to natural cooling.PBS cleans 5 minutes * 3 times, drips 5%BSA confining liquid, is placed in wet 37 ℃ of incubators of box and hatches 1 minute.Suck tissue unnecessary liquid around, drip PBS liquid dilution primary antibodie Fos (1:50) or TH (1:50) or primary antibodie DBH (1:100), hatch after 1 hour dislocation in 4 ℃ of refrigerator overnight for 37 ℃.PBS liquid cleans 5 minutes * 3 times.Drip biotinylation two anti-, in 37 ℃, hatch 1 hour.PBS cleans 5 minutes * 3 times.Blot PBS, drip SABC, hatch 1 hour for 37 ℃.PBS cleans 5 minutes * 3 times.Drip DAB developer, examine under a microscope and control chromogenic reaction, use distilled water cessation reaction in good time.Haematoxylin is redyed; Hydrochloride alcohol differentiation, tap water returns indigo plant; Conventional gradient ethanol (75%～100%) dewaters, dimethylbenzene is transparent, neutral gum mounting.Under microscope, blind method is observed ImmunohistochemistryResults Results, under high power lens (* 40), count respectively under each visual field, 3, district of large brain striatum brain Fos-positive cell number and DBH-positive neuron cell number (Yu under TH-positive neuron cell number and the visual field, 3, prefrontal cortex brain district, etal., 2013).Result represents with mean ± standard deviation, and the significant difference between SPSS16.0 software one-way ANOVA analysis bank, the results are shown in Figure 3～4, and statistical result is as shown in table 11:
The impact of table 11 pharmaceutical composition provided by the invention on BDH, TH and Fos in 6-OHDA neonate rat ADHD model cerebral cortex
* show and the blank group of P<0.05 that compares, * * shows and the blank group of P<0.01 that compares
As shown in table 11; compare with blank group; negative control group BDH, TH, Fos express significantly increases (P<0.01); with independent use tomoxetine hydrochloride; expression to appeal three has no significant effect; and significantly (P<0.01) rise brain DBH, TH and Fos expression of pharmaceutical composition provided by the invention; prompting taurine or taurine and tomoxetine coupling have protective effect to the noradrenergic neuron of ADHD rat and dopaminergic neuron, and two medicine couplings produce obvious synergistic function.
Embodiment 3 pharmaceutical composition pharmacokinetics provided by the invention and brain distribute and study
According to < < medicine registration management way > > and the requirement of < < chemical drugs Non-clinical Pharmacokinetics research guideline > > related content; take SD rat as study subject, pharmacokinetics and brain distribution preliminary study to tomoxetine hydrochloride after alone and coupling taurine.
Get 10 of SD rats and be divided at random bis-groups of A, B, before experiment, fasting is 12 hours.A group single oral gives tomoxetine hydrochloride, and dosage is 5mg/kg; B group single oral is combined and is given tomoxetine hydrochloride and taurine, and dosage is respectively 5mg/kg, 0.9g/kg.In administration process, guarantee that SD rat takes medicine completely.Gather blank blood before administration, after administration, in 0.033,0.083,0.25,0.5,1,2,3,5,7,9,12 and 24h tail vein blood 0.3ml, blood sample is put in heparinization blood taking tube, the centrifugal 5min of 8000rpm, and separated plasma, to be measured in-40 ℃ of Refrigerator stores.
Separately get 18 of SHR rat models and be divided at random G, the large group of H two, before experiment, fasting is 12 hours.G group single oral gives tomoxetine hydrochloride, and dosage is 5mg/kg; H group single oral is combined and is given tomoxetine hydrochloride and taurine, and dosage is respectively 5mg/kg and 0.9g/kg.After administration 0.5,3 and 7h sacrificed by decapitation rat, three rats of each time point.In 1 minute, divide and get cerebral tissue, with normal saline drip washing surface, filter paper blots, and weighs, and puts in homogenizer, by cerebral tissue: normal saline=1:1 (m/m), adds normal saline, homogenate.It is to be measured that homogenate is put-40 ℃ of Refrigerator stores.
Adopt LC/MS/MS method to measure tomoxetine hydrochloride and the content of taurine in each sample, plasma sample room temperature is thawed, and the accurate 100 μ l blood plasma of drawing are in 1.5ml centrifuge tube; add inner mark solution (768ngmL-1) 10 μ l; mobile phase 10 μ l, formic acid 20 μ l, vortex 1min mixes.Add ethyl acetate: dichloromethane (4:1) 1ml, vortex 3min mixes, the centrifugal 5min of 1200rpm, draw supernatant to tool plug centrifuge tube, 37.5 ℃ of Nitrogen evaporators dry up, and residue redissolves by 100 μ l mobile phases, vortex 1min, the centrifugal 5min of 1200rpm, gets supernatant 20 μ l sample introductions.
Brain tissue sample's same treatment.
Detection method is:
Chromatographic condition
Mobile phase: methanol (0.2% formic acid): water (containing 0.02mol/l ammonium acetate) (80:20, V/V);
Chromatographic column: Inertsil ODS-3 (100 * 2.1mm, 5 μ m);
Flow velocity: 300 μ l/min;
Column temperature: 30 ℃;
Sample size: 20 μ l;
Mass spectrum condition
Ion detection mode (Scan Type): MRM;
Ion polarity (Polarity): positive;
Ionizing mode: ESI;
The distribution testing result of medicine in cerebral tissue and blood plasma is as shown in table 12:
Distribution in table 12 tomoxetine hydrochloride cerebral tissue and plasma sample
Table 12 result shows: SD rat single oral tomoxetine hydrochloride gives after tomoxetine hydrochloride and taurine with combining, and by LC/MS/MS method, measures tomoxetine hydrochloride concentration in cerebral tissue.After being arranged, the data obtained obtains the scattergram of tomoxetine hydrochloride in SD rat brain in Figure 12.From chart, after drug combination, at 0.25h, 0.5h, 2h, 3h, during 5h, in SD rat cerebral tissue, the concentration of tomoxetine hydrochloride is all greater than the concentration of tomoxetine hydrochloride in individually dosed HouSD rat cerebral tissue.During this presentation of results drug combination, taurine may promote tomoxetine hydrochloride to see through blood brain barrier to enter cerebral tissue.
Medicine dynamic experiment result shows: single oral tomoxetine hydrochloride gives after tomoxetine hydrochloride and taurine with combining, the AUC0-T of tomoxetine hydrochloride is respectively 129.17 ± 39.637 μ ghL-1 and 128.96 ± 46.422 μ ghL-1AUC0-∞ are respectively 140.608 ± 41.383 μ ghL-1 and 142.661 ± 44.445 μ ghL-1, Cmax is respectively 65.537 ± 34.847ngmL-1 and 128.96 ± 46.422ngmL-1, Tmax is respectively 0.307 ± 0.197 and 0.363 ± 0.405h, T1/2 is respectively 2.942 ± 0.706h and 3.378 ± 1.666h.
Epidemiological Analysis by statistics, AUC0-T, AUC0-∞, the T of tomoxetine hydrochloride during individually dosed and drug combination
maxno difference of science of statistics (P>0.05), C
maxthere is significant difference (P<0.05), the C of tomoxetine hydrochloride after administering drug combinations
maxbe significantly less than the C after individually dosed
max.Result shows that drug combination compares with individually dosed, and the degree of absorption of tomoxetine hydrochloride in SD rat body is without significant difference.
The preparation of the medicine of embodiment 4 treatment hyperkinetic syndrome provided by the invention
Take 100 grams of tomoxetine hydrochloride, 100 grams of taurines, 300 grams, mannitol, 100 grams of starch, 3 grams of magnesium stearate, 2 grams of micropowder silica gels, fully mix, add appropriate distilled water to stir and granulate, cross 20 mesh sieves, dry, tabletting, every agreement that contracts a film or TV play to an actor or actress 0.3g.
The preparation of the medicine of embodiment 5 treatment hyperkinetic syndrome provided by the invention
Take 200 grams of tomoxetine hydrochloride, 150 grams of taurines, 550 grams, mannitol, 150 grams of starch, 5 grams of magnesium stearate, 3 grams of micropowder silica gels, fully mix, add appropriate distilled water to stir and granulate, cross 20 mesh sieves, dry, tabletting, every agreement that contracts a film or TV play to an actor or actress 0.3g.
The preparation of the medicine of embodiment 6 treatment hyperkinetic syndrome provided by the invention
Take 200 grams of tomoxetine hydrochloride, 100 grams of taurines, 325 grams, mannitol, 125 grams of starch, 5 grams of magnesium stearate, 3 grams of micropowder silica gels, fully mix, add appropriate distilled water to stir and granulate, cross 20 mesh sieves, dry, pack, every bag of about 0.3g.
The preparation of the medicine of embodiment 7 treatment hyperkinetic syndrome provided by the invention
Take 4 grams of tomoxetine hydrochloride, 200 grams of taurines, 315 grams, mannitol, 130 grams of starch, 4 grams of magnesium stearate, 2.5 grams of micropowder silica gels, fully mix, add appropriate distilled water to stir and granulate, cross 20 mesh sieves, dry, the about 0.3g of fill capsule.
The medicine effect of embodiment 8 treatment hyperkinetic syndrome provided by the invention detects
Laboratory animal: select 18 of healthy male SHR rats, be 10 weeks age in week, and body weight 265～295g, purchased from Sichuan Academy of Medical Sciences institute of lab animals.
Grouping and administration: 18 male SHR rats are divided into 3 groups at random, every group 6, gastric infusion is respectively experimental group, positive controls and blank group, wherein, experimental group gives the medicine that embodiment 2 provides, and positive controls gives tomoxetine hydrochloride, and blank group gives isopyknic normal saline.Dosage is all 10mg/kg, and every morning gavages, and 1 times/day, gavages continuously 14 days.Experimental session is given every rat 15～20g feedstuff every day, and water can freely obtain.
Detection method: above-mentioned each group is unified to draw materials in continuous gavage for 14 days afterwards, first use 10% chloral hydrate intraperitoneal injection of anesthesia rat, then by rat sacrificed by decapitation, on ice platform, get rapidly brain and isolate Hippocampus and cortex of frontal lobe tissue, after being weighed respectively, it with tinfoil, wraps, be stored in-70 ℃ of refrigerators, then high-efficient liquid phase technique detects norepinephrine (NE), 5-hydroxy tryptamine (5-HT) content.
Statistical method: use SPSS11.5 software kit to carry out data compilation and statistical analysis.Measurement data variance neat person check with t, and one factor analysis of variance is relatively used in heterogeneity of variance person rank test between group.Result is as shown in table 13:
The medicine effect of table 13 treatment hyperkinetic syndrome provided by the invention detects
| Group | Methylepinephrine (NE) | 5-hydroxy tryptamine (5-HT) |
| Experimental group | 23.65±8.69 ** | 12.24±5.63 ** |
| Positive controls | 45.24±3.21 ** | 9.24±13.92 * |
| Blank group | 34.84±8.63 | 6.47±12.65 |
* show and the blank group of P<0.05 that compares, * * shows and the blank group of P<0.01 that compares
Result shows: compare with blank group, the tablet that embodiment 2 provides significantly (P<0.01) reduces in SHR rat brain the content of NE in hippocampal tissue, the content of 5-HT in rising cortex of frontal lobe tissue, can significantly raise the two content of positive control medicine, NE (P<0.01) wherein, 5-HT (P<0.05).
Below be only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a pharmaceutical composition, is characterized in that, comprising: tomoxetine hydrochloride and taurine.
2. pharmaceutical composition according to claim 1, is characterized in that, the mass ratio of described tomoxetine hydrochloride and taurine is 1:0.5～50.
3. pharmaceutical composition as claimed in claim 1 application in SOD accelerative activator within preparing brain.
4. pharmaceutical composition as claimed in claim 1 application in MDA inhibitor within preparing brain.
5. the application in the medicine of pharmaceutical composition as claimed in claim 1 GSH content in preparation increases brain.
6. the application in the medicine that pharmaceutical composition as claimed in claim 1 DBH, TH or Fos in preparation promotes brain expresses.
7. the application of pharmaceutical composition as claimed in claim 1 in the medicine of preparation treatment hyperkinetic syndrome.
8. a medicine for the treatment of hyperkinetic syndrome, is characterized in that, comprises pharmaceutical composition and pharmaceutically acceptable adjuvant as described in claim 1～2 any one.
9. medicine according to claim 8, is characterized in that, described pharmaceutically acceptable adjuvant is excipient, filler and lubricant.
10. medicine according to claim 8, is characterized in that, the mass fraction of wherein said tomoxetine hydrochloride is 2%～50%; The mass fraction of described taurine is 2%～50%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001026642A2 (en) * | 1999-10-08 | 2001-04-19 | Joyce Corinne Bechthold | Methods and compositions for treating neurobehavioral disorders |
| US20050209303A1 (en) * | 2004-03-22 | 2005-09-22 | Gottfried Kellermann | Method of treatment for high neurotransmitter pattern |
| WO2012178014A1 (en) * | 2011-06-24 | 2012-12-27 | K-Pax Pharmaceuticals, Inc. | Compositions and methods for treatment of chronic fatigue |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001026642A2 (en) * | 1999-10-08 | 2001-04-19 | Joyce Corinne Bechthold | Methods and compositions for treating neurobehavioral disorders |
| US20050209303A1 (en) * | 2004-03-22 | 2005-09-22 | Gottfried Kellermann | Method of treatment for high neurotransmitter pattern |
| WO2012178014A1 (en) * | 2011-06-24 | 2012-12-27 | K-Pax Pharmaceuticals, Inc. | Compositions and methods for treatment of chronic fatigue |
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| Title |
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| 杜亚松: "治疗ADHD的新药-托莫西汀", 《中国儿童保健杂志》, vol. 16, no. 1, 29 February 2008 (2008-02-29) * |
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