CN103142996B - Application of endogenic marihuana peptide agonist (m) VD-Hp alpha in preparing analgesics - Google Patents

Abstract

The invention discloses application of endogenic marihuana peptide agonist (m) VD-Hp alpha in preparing analgesics, which means that an injection preparation or lyophilized powder preparation is prepared from the endogenic marihuana peptide agonist (m) VD-Hp alpha serving as an active ingredient and auxiliary materials according to a conventional process of pharmaceutics. A series of in-vivo activity detections indicate that marihuana endogenous peptide ligand (m) VD-Hp alpha has strong central analgesia activity, and has an advantage of low central side effect, which means that the marihuana endogenous peptide ligand (m) VD-Hp alpha seldom has the side effects of constipation and hypokinesia in an effective analgesic dose range, has a small influence on body temperature, and is slow in the analgesic tolerance process. Therefore, the endogenic marihuana peptide agonist (m) VD-Hp alpha has a good application prospect in preparing high-efficiency low-side-effect analgesics.

Description

Endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α is preparing the application in analgesic

Technical field

The invention belongs to technical field of biochemistry, relate to the application of a kind of endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α in prepared by efficient, lower pair acting analgesic thing.

Background technology

Chronic and control that is severe pain is people's difficult medical problem urgently to be resolved hurrily always.Existing various medicaments is used for the treatment of diversified pathological state at present, and this is comprising (Drugs 2007,2121:2133) such as opium, non-sterols anti-inflammatory agent, anticonvulsant, antidepressants, KETs.But these side effects of pharmaceutical drugs limit their clinical medicine dose and reduce its therapeutic effect.Except the analgesic mechanism of the pathomechanism and qualification medicine itself of studying chronic pain further, clinically more it is desirable that effectively, the novel drugs for the treatment of chronic pain that is nontoxic and that do not have maincenter side effect.

With a long history as clinical application of Fructus Cannabis.In Europe, Fructus Cannabis is just used to treat pain, spasm, asthma, sleep disordered, depressed and inappetence as far back as 19th-century.ISUZU company before twentieth century, almost being abrogated in treatment of Fructus Cannabis, partly cause be people fubaritic go out the chemical constitution of cannabinoid composition.Until 1964, the main component Δ of cannabinoid ⁹-THC is through spatial chemistry identified (Mult Scler. 2004,434:441).And then, find that the Fructus Cannabis system of body self comprises specific receptor and endogenous ligands, just start the further investigation (Dtsch Arztebl Int. 2012,495:501) having endogenous Fructus Cannabis system and clinical practice thereof.Existing research shows that Fructus Cannabis all has important regulating action in motion, the pain sensation, body temperature, learning and memory and cardiovascular.Some cannabis parts have been widely used for antiinflammatory, sedation and analgesia clinically, and achieve good efficacy in the diseases such as treatment vomiting, inappetence, glaucoma, bronchial asthma, senile sclerosis of blood vessels.Research in recent years also shows that cannabis part all has potential application prospect in fat-reducing, ischemia resisting oxygen injury, antitumor, epilepsy, treatment nerve retrograde affection etc.

Fructus Cannabis most important function in nervous system is eased pain exactly (CNS Neurol Disord Drug Targets. 2009,8:403), but existing result of study finds that cannabis analgesic used at present limits it and extensively uses due to their side effect.As Fructus Cannabis easily produces analgesia tolerance when treating pain, in addition, also easily produce that hypokinesia, movement disorder and extremity are stiff, the side effect (Pharmacol. Rev. 2006,58:389) such as constipation and addiction.In addition, Fructus Cannabis can also cause cognitive disorder, the side effect such as cardiopalmus, postural hypotension, xerostomia, fertility go down, immunosuppressant.

Before, generally believe that Cannabinoids mainly comprises three major types: the Fructus Cannabis of extracting from natural plants, the Fructus Cannabis of synthetic and endogenous lipid Fructus Cannabis.The cannabis agonist found and identify and antagonist are mainly the cannabinol compounds (Pharmacol. Rev. 2006,58:389) of synthetic.The nineties in last century, first find some endogenous lipid molecules can in conjunction with and act on cannabinoid receptors, be called as endogenous lipid ligand molecular, mainly comprise N-anandamide (AEA) and 2-AG (2-AG).These endogenous Fructus Cannabis lipid part and relevant enzymes thereof, and cannabinoid receptors CB ₁and CB ₂together constitute endogenous Fructus Cannabis system (Pharmacol. Rev. 2006,58:389).

In recent years, from the endonuclease bamhi of hemoglobin alpha-chain and β chain, some have been found respectively to CB ₁and CB ₂have different affinitys and optionally montage fragment, they show as different pharmacological characteristics such as the antagonist of Fructus Cannabis, inverse agonist and agonist etc.M () VD-Hp α is the endonuclease bamhi of the hemoglobin alpha-chain in mice source, be the structure of 11 peptides, its structure sequence is: Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH.Its structural formula is as follows:

Existing vitro functional test experience shows that (m) VD-Hp α is CB ₁the selection agonist (FASEB J. 2009,3020:3029) of receptor.Further signal transduction pathway found that, (m) VD-Hp α is by CB ₁the receptor-mediated signal transduction pathway being different from existing Fructus Cannabis part, can cause Ca in ERK phosphorylation level or cell ²⁺the increase (FASEB J. 2009,3020:3029) of release.

Summary of the invention

Main purpose of the present invention is to provide that endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α is efficient in preparation, application in lower pair acting analgesic thing.

Below by experiments in vivo, the pharmacological effects of (m) VD-Hp α in the pain sensation, motion, body temperature and gastrointestinal motility adjustment in the present invention is described.

1, the analgesic experiment of (m) VD-Hp α

After the injection of tricorn and spinal levels, mice photo-thermal tail-flick test is utilized to carry out the Activity determination of pain sensation regulating action in body and compared.

(1) the tricorn administration of mice

The horizontal pain sensation test experience of tricorn, with mice, need carry out the tricorn pipe laying operation of mice, in advance to ensure the accuracy in drug injection site.Brain solid positioner is utilized to carry out the tricorn pipe laying of mice.Kunming system male mice, body weight 21 ± 2 g; Brain solid positioner is gulf, river I type.Mouse head field of operation, with after pentobarbital sodium anesthesia (i.p, 80mg/kg Mouse Weight), is cut off hair by mice, is placed in after brain solid positioner is fixed, field of operation iodophor disinfection, cuts scalp, expose skull, find bregma position along sagittal suture.From bregma 3 mm backward, to left/right 1 mm, be the top position of tricorn.Self-control stainless steel tube (24-gauge one section, meet a bit of PE-10 above and manage) inserts 3 mm downwards by above-mentioned position, is tricorn.One section of rustless steel string (28-gauge) is inserted in above-mentioned steel pipe, and to prevent, cerebrospinal fluid is excessive and external impurities infects and blocking steel pipe.Steel pipe is fixed with medical courses in general dental base acrylic resin powder and glue, after solidifying, sew up wound.Post operation, mice recovers 4 days, and the 5th day starts subsequent experimental.The operating theater instruments related in experiment, stainless steel tube etc. all need to sterilize before surgery.Medicine dissolution, in normal saline ,-20 DEG C of preservations, thaws before using.

(2) intrathecal injection of mice

Spinal levels administration adopts conscious mouse intrathecal injection method, the method (Eur. J. Pharmacol. 1980,67:313) that concrete operations have been reported with reference to Hylden and Wilcox.25 μ l microsyringes are directly inserted into the subarachnoid space between L5 and L6.When arachnoidea is punctured along with mice fierce and significantly whipping action or tail be serpentine, normal saline and medicine are injected in subarachnoid space with the speed of 5 μ l/10 seconds.

(3) pain sensation test experience

Use Kunming system male mice, utilize photo-thermal whipping instrument to carry out the impact of detection of drugs on pain sensation effect.Ambient temperature controls at 22 ± 1 DEG C, and laboratory animal can ad lib, drinking-water.Mice tricorn per injection 4 μ l medicine.First measure basic TFL (3-5 second) before administration, too responsive or blunt mice is discarded.The TFL of the 5th, 10,15,20,30,45,60 minutes after record administration.For preventing scalding, be set to the longest TFL by 10 seconds, the whipping time all came in 10 seconds more than 10 seconds.

(4) MPE and EC of medicine analgesic effect is calculated ₅₀value

Pain sensation regulating action utilizes maximum possible effect MPE(maximum possible effect) value evaluates, MPE(%)=100 × [(threshold of pain-Basic Pain Threshold after administration)/(10 seconds-Basic Pain Threshold)].Relevant MPE data represent with mean+/-standard error (Means ± S.E.M.), and the group difference one factor analysis of variance (the Dunnett inspection of one-way ANOVA) of different pharmaceutical concentration process carries out the statistics and analysis of data, ^{* *}p < 0.001 represents to there is significant difference between the MPE value of drug treating group and normal saline group.Experimental result is shown in Fig. 1-2.

EC ₅₀value expression medicine produces drug dose during 50% maximum analgesic effect.Utilize statistics software GraphPad Prism 5.0 version, depict the amount effect curve (see figure 3) of (m) VD-Hp α MPE value of the maximum analgesic effect time point of two kinds of different dosing levels in tricorn, sheath respectively, and calculate their EC in difference injection level ₅₀value and 95% confidence interval.Relevant experimental data is shown in Table 1.

The analgesic activity that table 1 mice varying level injection (m) VD-Hp α produces

Normal saline group is blank solvent matched group, and the drug level of intracerebroventricular injection (m) VD-Hp α is respectively 3.75,7.5,15 and 22.5 nmol.Every treated animal number is 7-8.As shown in Figure 1, mice intracerebroventricular injection (m) VD-Hp α can produce analgesic activity in dose-dependant ground.M () VD-Hp α can continue 60 minutes at the analgesic activity of the above level of spinal cord, and 15 minutes after drug injection reach maximum analgesic effect.

Equally, the analgesic activity that intrathecal injection (m) VD-Hp α (1.8,3.75,7.5 and 15 nmol) can cause dose-effect to comply with, reaches the strongest for after this acts on drug injection 10 minutes, and can continue 45 minutes (as shown in Figure 2).

As shown in Fig. 1-2 and table 1, (m) VD-Hp α has stronger central analgesia active under the injection level that tricorn, spinal cord are different.Further, (m) VD-Hp α analgesic activity of spinal levels is better than the above level of spinal cord.

2, the analgesia tolerance experiment of (m) VD-Hp α

Mice tricorn continuous eight days injectable drugs, utilize photo-thermal tail-flick method to detect their analgesia tolerance phenomenon, inquire into the pharmacological activity of compound (m) VD-Hp α of the present invention in analgesia tolerance further.

(1) pain sensation tolerance test method of mice

Kunming system male mice, the continuous injectable drug of tricorn eight days, injection every day once, utilizes photo-thermal whipping instrument to carry out detection of drugs and injects impact on pain sensation effect continuously.Ambient temperature controls at 22 ± 1 DEG C, and laboratory animal can ad lib, drinking-water.Mice tricorn per injection 4 μ l medicine.Measure basic TFL (3-5 second) before first day drug injection, too responsive or blunt mice is discarded.The TFL of the maximum analgesic effect time point of related drugs after recording administration in experiment, some writing time as intracerebroventricular injection (m) VD-Hp α is after administration 15 minutes; The time measuring point of blank solvent matched group and being consistent of (m) VD-Hp α.For preventing mousetail from scalding, be set to the longest TFL by 10 seconds.

(2) tolerance test data statistics

Experimental data MPE(maximum possible effect) represent, MPE(%)=100 × [(threshold of pain-Basic Pain Threshold after administration)/(10 seconds-Basic Pain Threshold)].The analgesia tolerance effect of medicine utilizes the MPE value of the maximum analgesic effect time point of related drugs to compare.MPE data mean+/-standard error (Means ± S.E.M.) represents, the difference one factor analysis of variance (the Bonferroni inspection of one-way ANOVA) of mice eight days analgesic activities is added up, ^{* *}p < 0.001 represents there is significant difference compared with first day (m) VD-Hp α analgesic activity.Experimental result as shown in Figure 4.

Normal saline group is blank solvent matched group, and the drug dose of intracerebroventricular injection (m) VD-Hp α is 2 × EC ₅₀value and 3 × EC ₅₀value, i.e. 13.4 and 20.1 nmol.The experimental data of Fig. 4 shows, after mice tricorn continuous eight days injection blank solvent contrast normal saline, mice does not produce analgesic activities.Compared with contrasting normal saline group with blank solvent, (m) VD-Hp α of the various dosage of first day intracerebroventricular injection extends the TFL of mice significantly.When mice tricorn injects (m) VD-Hp α eight days continuously, the 5th day analgesic activity MPE value just starts to decline significantly upon administration, and continues to the 8th day.Compared with first day, the diversity of the 5th day to the 8th day is extremely remarkable.But (m) VD-Hp α analgesia tolerance process is comparatively slow, just starts to have occurred analgesia tolerance phenomenon, and still maintained the analgesic activity (compared with first day) of about 50% at the 8th day from the 5th day.

Cannabis analgesic, except tolerating, can also cause and suppress motion, side effect such as reduction body temperature and constipation etc.Therefore, further evaluation has been done to (m) VD-Hp α regulating action in these areas.By mice intracerebroventricular injection medicine, with the side effect of different detection method evaluation (m) VD-Hp α in potent analgesics weight range.

3, the detection of (m) VD-Hp α locomotor activity regulating action

(1) mice is in body locomotor activity detection method

Kunming system male mice, body weight 20 ± 2 g.Tricorn pipe laying uses water maze tracing system (Chengdu Tai Meng scientific & technical corporation) to carry out locomotor activity mensuration after recovering 4 days.Put into detection box (50 cm x 50 cm x 30 centimetres) after every mice intracerebroventricular injection medicine or saline, record the distance of horizontal movement in 15 minutes.

(2) add up in body locomotor activity experimental data

Locomotor activity experimental data is evaluated with the horizontal movement distance that mice is total.The relevant data mean+/-standard error (Means ± S.E.M.) often organizing the total distance of mouse movement represents, the group difference one factor analysis of variance (the Bonferroni inspection of one-way ANOVA) of different pharmaceutical concentration process carries out data statistics and analysis ^{* *}p < 0.001 represents to there is significant difference between the locomotor activity of drug treating group and normal saline group.Experimental result as shown in Figure 5.

In Figure 5, normal saline group is blank solvent matched group, and the drug level of (m) VD-Hp α is respectively 1 × EC ₅₀value, 2 × EC ₅₀value and 3 × EC ₅₀value, namely 6.7,13.4 and 20.1nmol, often organizing number of mice is 9.Experimental result shows, the total distance of motion of blank solvent matched group is 4724 ± 290 centimetres, compared with normal saline group, (m) VD-Hp α (6.7 and 13.4 nmol) of mice intracerebroventricular injection low dosage, on the locomotor activity of mice almost without impact; Only have when injecting high dose (m) VD-Hp α (20.1nmol), the total distance of motion of mice is 940 ± 193 centimetres, inhibits the locomotor activity of mice significantly.Therefore, (m) VD-Hp α only has the side effect just occurring suppressing motion during high dose.

4, the detection of (m) VD-Hp α thermoregulation effect

(1) the thermoregulation detection method of mice

Kunming system male mice, body weight 27 ± 2 g.Tricorn pipe laying recovers after 4 days for the determination of activity of medicine thermoregulation.Room temperature 22 ± 1 ° of C, experimental period is between 10:00-14:00.Be fixed to parallel for mouse tail on mouse fixing.Temperature sensor is inserted mice internal rectum 2.5 centimetres, use BL-420E ⁺type Physiological Experiment system log (SYSLOG) mouse temperature.After every mice intracerebroventricular injection medicine or saline, the mouse temperature of record 10,20,30,40,50 and 60 minutes points.

(2) body temperature experimental data statistics

Body temperature experimental data is evaluated with the body temperature value of each time point of mice.The relevant data mean+/-standard error (Means ± S.E.M.) of the body temperature often organizing each time point of mice represents, the group difference one factor analysis of variance (the Bonferroni inspection of one-way ANOVA) of different pharmaceutical concentration process carries out data statistics and analysis ^*p < 0.01 He ^{* *}p < 0.001 represents to there is significant difference between the body temperature of drug treating group and normal saline group.Experimental result as shown in Figure 6.

In figure 6, normal saline group is blank solvent matched group, and the drug level of (m) VD-Hp α is respectively 1 × EC ₅₀value, 2 × EC ₅₀value and 3 × EC ₅₀value, namely 6.7,13.4 and 20.1nmol, often organizing number of mice is 9.Experimental result shows, medicine is at intracerebroventricular injection after 10 minutes, the most remarkable to the reduction effect of body temperature.Now, the body temperature of saline control group mice is-0.06 ± 0.06 DEG C, and compared with normal saline group, (m) VD-Hp α of mice intracerebroventricular injection low dosage 6.7 nmol, on the body temperature of mice almost without impact (-0.59 ± 0.11 DEG C); Only have when injecting moderate or high dose (m) VD-Hp α (13.4 and 20.1nmol), the body temperature of mice is respectively-1.07 ± 0.12 DEG C and-1.64 ± 0.22 DEG C, significantly reduces the body temperature of mice.Therefore, (m) VD-Hp α is at 1 × EC ₅₀value, 2 × EC ₅₀on the impact of body temperature less (being respectively-0.6 DEG C and-1.1 DEG C) under dosage; Only when high dose, (m) VD-Hp α just has cooling effect significantly.

5, the detection of (m) VD-Hp α gastrointestinal motility regulating action

(1) mice in body gastrointestinal motility detection method

Kunming system male mice, body weight 20 ± 2 g.Tricorn pipe laying recovers to detect for gastrointestinal motility after 4 days.Detect at body gastrointestinal motility and select conventional carbon dust detection method (Peptides, 2000,21:295).Concrete experimentation is: mice fasting 16 hours (can arbitrarily drink water during fasting) before gastrointestinal motility experiment, then intracerebroventricular injection medicine, was filled into stomach by preprepared active carbon suspension (a kind of normal saline suspension containing 5% active carbon and 10% Radix Acaciae senegalis) with the volume per os of every 10 grams of body weight 0.1 ml after 15 minutes.After active carbon suspension pours into 30 minutes, cervical dislocation puts to death animal, then carefully takes out the animal small intestinal from pylorus to caecum.Measure the total small intestinal length of pylorus to caecum and the maximum distance of active carbon suspension movement.

(2) add up in body gastrointestinal motility experimental data

Gastrointestinal motility experimental result gastrointestinal motility percentage ratio is evaluated, and the gastrointestinal motility percentage ratio of every mice is calculated divided by the percentage ratio after total small intestinal length by active carbon suspension displacement.Relevant data represent with the mean+/-standard error (Means ± S.E.M.) of gastrointestinal motility percentage ratio, and the group difference one factor analysis of variance (the Bonferroni inspection of one-way ANOVA) of different pharmaceutical concentration process carries out data statistics and analysis.Experimental result as shown in Figure 7.

In the figure 7, normal saline group is blank solvent matched group, and the drug level of (m) VD-Hp α is respectively 1 × EC ₅₀value, 2 × EC ₅₀value and 3 × EC ₅₀value, namely 6.7,13.4 and 20.1nmol, often organize number of mice be 8-9 only.Experimental result shows, the gastrointestinal motility percentage ratio of blank solvent matched group is 76%, and the gastrointestinal motility percentage ratio of (m) VD-Hp α of intracerebroventricular injection 6.7,13.4 and 20.1nmol is respectively 67%, 78% and 76%.Compared with normal saline group, (m) VD-Hp α on the gastrointestinal motility of mice almost without impact; Therefore, in potent analgesics weight range, (m) VD-Hp α is without the side effect of constipation.

In sum, in the present invention, Fructus Cannabis endogenous peptide part (m) VD-Hp α has stronger central analgesia activity, and there is the low advantage of maincenter side effect, namely in potent analgesics weight range, substantially without the side effect of constipation and hypokinesia, little to temperature influence, and analgesia tolerance process is slow.Therefore, endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α has potential using value at efficient, lower pair acting analgesic thing in preparing.

The pharmaceutical preparation of general solution state needs to add a large amount of salinities, antiseptic and stabilizing agent, and its pH changes very easily in time, cause its difficult problem in preservation, and the adding of antiseptic and stabilizing agent, the toxic and side effects of medicine itself is increased, greatly limit medicine application clinically.And the dissolubility of analgesic (m) VD-Hp α of the present invention is fabulous, reverse high performance liquid chromatography (HPLC) C can be utilized ₁₈high-purity (m) the VD-Hp α sample preparation of column separating purification becomes the preparation of two kinds of forms, i.e. injection and freeze-dried powder.M () VD-Hp α ejection preparation is easy to use, fast, be by formulated in water-soluble for (m) VD-Hp α lyophilized powder, the solution such as physiology isotonic saline solution and glucose; And the freeze-dried powder relative injection preparation of (m) VD-Hp α, the shelf-life is longer, it is more convenient to transport, and is easy to preserve.

Accompanying drawing explanation

The Time-activity-curve of the dose dependent analgesic activity that Fig. 1 produces for mice intracerebroventricular injection (m) VD-Hp α;

The Time-activity-curve of the dose dependent analgesic activity that Fig. 2 produces for mice intrathecal injection (m) VD-Hp α;

The amount effect curve of the analgesic activity that Fig. 3 mice tricorn and intrathecal injection (m) VD-Hp α produce;

The change of the analgesic activity caused by the above horizontal continuity of Fig. 4 mouse spinal cord eight days intracerebroventricular injection (m) VD-Hp α;

Fig. 5 mice intracerebroventricular injection (m) VD-Hp α is on the impact of its locomotor activity;

Fig. 6 mice intracerebroventricular injection (m) VD-Hp α is to the regulating action of its body temperature;

Fig. 7 mice intracerebroventricular injection (m) VD-Hp α is on the impact of its gastrointestinal motility.

Detailed description of the invention

Below by specific embodiment, the synthetic method of Fructus Cannabis peptide excitomotor (m) VD-Hp α of the present invention and the preparation of preparation are described further.

The synthesis of embodiment one, Fructus Cannabis peptide excitomotor (m) VD-Hp α

I material

Instrument: the Delta 600 that high performance liquid chromatograph (HPLC) is Waters company; Analytical column: DELTA PAK 5 μ C18 300 3.9 × 150mm; Preparative column: DELTA PAK 15 μ C18 300 7.8 × 300mm.Mass spectrograph is PE Biosystems, Mariner System 5074.Manual solid-phase Peptide synthesizer, (the 14th page of Fig. 4 in concrete " the Fmoc solid phase peptide synthesis " compiled with reference to Chen WC and White PD of the design principle of synthesizer is made by glazier by after this laboratory design, and done part improvement on its basis, namely substitute drum nitrogen method in order to mechanical agitation mode, thus reach the well-mixed object of reaction solution).Reagent: resin is Fmoc-His (Trt)-Wang-Resin(1% DVB, 200 ~ 400 mesh, substitution value S=0.57 mmol/g resin), purchased from Tianjin Nankai with become company. nthe aminoacid (Fmoc-Aa) that-α-Fmoc protects, n-hydroxy benzo triazole (HOBt), O-BTA- n, N, N', N'-tetramethylurea-hexafluorophosphate (HBTU), diisopropylethylamine (DIEA) and tri isopropyl silane (TIS) are purchased from the biochemical (Shanghai) Co., Ltd. of gill.1,2,3-indantrione monohydrate is Shanghai reagent three factory product.Dichloromethane (DCM), n,N-dimethyl formamide (DMF), hexahydropyridine (piperidines), methanol (MeOH) and pyridine are all purchased from Tianjin second chemical reagent work, and trifluoroacetic acid (TFA), phenol and pyridine are Tianjin reagent one factory product; Above organic reagent all processes through overweight steaming before using.

the thick peptide symthesis of (m) VD-Hp α

Adopt the solid phase polypeptide synthesis of Fmoc Preservation tactics.Peptide elongation adopts and connects peptide method (step by step) one by one.Concrete operation step is as follows:

(1) resin pretreatment: 1000 mg Fmoc-His (Trt)-Wang resins are added in synthesizer, then stir 30 min after the DCM adding 8 ml, make the abundant swelling rear decompressing and extracting solvent of resin.

(2) remove Fmoc radical protection: swelling, drain in the resin of solvent, add the solution of 8 ml hexahydropyridine: DBU:DMF=1:1:98, drain after stirring 5 min, drain after repeating 3 times.Finally add the DMF of 8 ml, drain after stirring 3 min, repeat 4 times, obtain the resin sample removing Fmoc radical protection.

(3) indenes inspection: add in the following order in test tube: resin sample, 0.1 ml reagent , 0.2ml reagent , 0.1 ml reagent , observe after boiling water bath 3-10 min.Solution, resin are blue expression Fmoc blocking group and remove completely.Formula for three kinds of reagent of indenes inspection is: reagent 80 g phenol/20ml ethanol; Reagent 0.001 M potassium cyanide (the water)/98ml pyridine of 2.0 ml; Reagent 5 g 1,2,3-indantrione monohydrate/100 ml ethanol.

(4) condensation: successively will with DMF in small beaker n-α-Fmoc blocking group aminoacid (Fmoc-Aa), n-hydroxy benzo triazole (HOBt), O-BTA- n, N, N', N'-tetramethylurea-hexafluorophosphate (HBTU) dissolves completely with the mol ratio of 1:1:1, then the diisopropylethylamine (DIEA) adding Fmoc-Aa doubling dose fully mixes afterwards, obtains mixed solution; Join in synthesizer by the resin removing Fmoc radical protection that 8 ml mixed solutions and step (2) obtain, under argon shield, stirring reaction 60 min, drains solvent.Add the DMF of 8 ml, drain after stirring 3 min, repeat 3 times, obtain peptide resin sample; Carry out indenes inspection according to step (3), indenes inspection solution is yellowish, resin is that colourless expression condensation is complete.

(5) prolongation of peptide chain: repeat step (2), (4) 7 times, according to peptide structure by the order of C-end to N-end, successively will be with nthe condensation one by one of-α-Fmoc blocking group aminoacid, after often step reaction completes, filters through suction function and removes reagent in reactor, until complete the condensation of the 9th amino acids residue.

(6) acetic anhydride end socket: after the 9th Pro condensation completes, with acetic anhydride, the Pro having neither part nor lot in reaction is closed.Add in resin peptide after getting the acetic anhydride of 50 times of resin mole and DIEA mix homogeneously, under argon shield, stirring reaction 30min, drains solvent; Use DMF repeated washing; Next step condensation reaction is carried out again, until last amino acid condensation completes after sloughing Fmoc protecting group by step (2).

m the peptide chain of () VD-Hp α is from the cutting resin

After the whole condensation of amino acid residue of peptide chain completes, according to operation in above-mentioned steps (2), last amino acid whose Fmoc group of peptide resin is removed completely.Then according to DCM 3 min × 2 time, MeOH 3 min × 1 time; DCM 3 min × 1 time, the operation of MeOH 3 min × 2 time alternately washes resin.Remove splash bar, by synthesizer sealing (plug), thoroughly drain (at least 2 hours).The peptide chain resin of drying is placed in reactor, adds the cutting agent (volume ratio is TFA:TIS: water=95:2.5:2.5) of 18ml, cleavage reaction 2.5 hours (stirring once for every 15 minutes, each stirring 1 minute) under room temperature.Filter, filtrate under not higher than the condition of 37 ° of C fully decompression be spin-dried for, then separate out precipitation with the ether higher than-10 ° of C, vibration makes thick peptide fully separate out with the form of white precipitate.By supernatant sucking-off after leaving standstill, the precipitation of the abundant solution modeling that adds water, is separated removing by ether with separatory funnel from aqueous phase, and merge aqueous phase obtains white thick peptide pressed powder 848.8mg by lyophilization, productive rate is 117 %.

the desalination of the thick peptide of (m) VD-Hp α and purification.

Be dissolved in by whole crude product in 20% acetic acid solution, solution crossed Sephadex G25 cross-linked dextran gel column (2.0 × 25cm), mobile phase is volumetric concentration 20% acetic acid solution.Ultraviolet Detector is utilized to collect main peak postlyophilization.Use reverse high performance liquid chromatography (HPLC) C again ₁₈post (XBridge TM BEH 130 C ₁₈, 19 mm × 250mm) and separation and purification is carried out to the peptide compounds of above-mentioned desalination, after being separated, collecting sample main peak, lyophilization, obtaining Fructus Cannabis peptide excitomotor (m) VD-Hp α) lyophilized powder.(m) VD-Hp α sample purity after purification is more than 98%, and the gross production rate of its synthesis is 58%.

The preparation of embodiment two, Fructus Cannabis peptide excitomotor (m) VD-Hp α ejection preparation.

Aseptic (m) VD-Hp α lyophilized powder 30mg be sub-packed in cillin bottle is dissolved in 5ml sterile physiological isotonic saline solution, abundant dissolving is afterwards in water white transparency, limpid aqueous solution, and experimentally aequum directly uses or uses with after isotonic saline solution dilution further.

The preparation of embodiment three, freeze-dried powder

Aseptically, by highly purified (m) VD-Hp α lyophilized powder 100 mg, by preparation of injection procedure operation, be sub-packed in 10 cillin bottles, jump a queue, roll lid sealing, obtain injection (m) VD-Hp α powder injection formulation of the present invention.

Claims (1)

1. endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α is preparing the application in analgesic, m () VD-Hp α is the endonuclease bamhi of the hemoglobin alpha-chain in mice source, be the structure of 11 peptides, its structure sequence is: Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH; With endogenous Fructus Cannabis peptide excitomotor (m) VD-Hp α for active component, be prepared into ejection preparation or freeze-dried powder by the common process of pharmaceutics.