CN1363275A - Application of paralytic PSP toxin in preparing antalgesic medicine - Google Patents
Application of paralytic PSP toxin in preparing antalgesic medicine Download PDFInfo
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Abstract
The application of paralytic PSP toxin in preparing antalgesic medicine is disclosed, which features that 5 of more than 20 PSP toxins are chosen, which are aminoformate toxin, N-sulfonylaminoformyl toxin deaminated farmyl toxin, deoxydeaminoformyl toxin and aminoformate-type N-hydroxy derivative. They can be applied individually or in conjunction with stupefacient analgesics, such as morphine, morphine methyl ether, etc. Its advantages include high curative effect, durable action and low toxic by-effect.
Description
(1) technical field:
The present invention relates to paralytic shellfish poison (Paralytic Shellfish Poisons, PSP) toxin is (hereinafter to be referred as the PSP toxin) new medicine use, specifically be that paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, it belongs to the application process technical field of Chemical composition that.
(2) technical background
Paralytic shellfish poison (PSP) toxin poisoning just has generation from ancient times.Nineteen twenty-eight, (J.Prevent.Med.2,365-394) once outstanding literary composition has described the history of relevant PSP toxin poisoning incident in detail to Meyer etc.But cause that really the poisoning that people pay attention to and further investigate betides nineteen twenty-seven, 102 people in about 50 mile range in north and south, California, USA Jinmen fall ill because of edible mussel.Through investigation, Meyer etc. think that some dinoflagellate is to make the toxigenous key factor of mussel.After this, people know gradually that just the PSP toxin has many types, and its source has nothing in common with each other.
Learn after deliberation: the PSP toxin is the derivant of a class tetrahydrochysene purine.The poisonous algae in main ocean that can produce the PSP toxin at present has: non-chain Alexander algae (Alexandrium acatenella), chain Alexander algae (A.catenella), thigh shape Alexandrium (A.cohorticula), Alexandrium tamarense (A.tamarense), chain unarmored dinoflagellate (Gymnodinium catenatum) etc.In addition, some kinds in antibacterial, cyanophyceae, the red algae (Jania sp.) also can produce the PSP toxin.In shellfish, snail, Eriocheir sinensis class, Fish, also once detected part PSP toxin.The PSP toxin that has been found that now has kind more than 20 (table 2), and the basic structure formula of PSP toxin is as follows:
Table 2 PSP toxin kind and title (Yu Rencheng, Zhou Mingjiang, Oceanologia et Limnologia Sinica 3,330-338 (1998))
| R1 | ??R2 | ??R3 | ??R4：????R4：????R4：????R4：????R4： ??????????????????OH??????????????H |
R1 wherein, R2, R3, R4 are four substituted radicals in the structural formula.
The PSP toxin is alkalescence, and the water solublity height dissolves in methanol, ethanol.More stable under the generic condition, but some groups wherein also can change, as C
11The spatial isomerismization of position hydroxy sulfonate group.A large amount of beta isomers that exist can be transformed into more stable αYi Gouti in the poisonous algae.Also this isomerization can take place in the shellfish tissue, the ratio of stable back isomer is tending towards α: β=3: 1.N-sulphonyl carbamyl toxoid can be taken off sulfonyl under heating, acid condition, generate corresponding carbamate toxoid; And at the corresponding deamination formoxyl of the next generation of stable condition toxoid.The PSP toxin belongs to the guanidine toxoid, and its active site is 7,8,9 a guanidine radicals, with valtage-gated Na
+The amino acid residue height in passage site 1 is affine, by selective exclusion Na
+In stream, hinder the formation of action potential and work.Each derivant of PSP toxin is to Na
+The blocking-up degree in passage site 1 has very big difference, and toxicity also has some differences (seeing Table 1).The toxicity of table 1 PSP toxin
| Toxin | Toxicity (MU/ μ mol) | Toxicity (STX Equal/ μ mol) | Toxicity (%) with respect to STX |
| ??GTX 5(B 1) | ????150 | ????27 | ????7 |
| ??GTX 6(B 2) | ????150 | ????27 | ????7 |
| ??C 1 | ????17 | ????＜1 | ????＜1 |
| ??C 2 | ????258 | ????47 | ????13 |
| ??STX | ????2100 | ????378 | ????100 |
| ??neoSTX | ????2300 | ????414 | ????110 |
| ??GTX 1 | ????1900 | ????342 | ????90 |
| ??GTX 2 | ????1000 | ????180 | ????48 |
| ??GTX 3 | ????1600 | ????288 | ????76 |
| ??GTX 4 | ????1900 | ????342 | ????90 |
| ??dcSTX | ????900 | ????162 | ????43 |
| ??dcneoSTX | ????900 | ????162 | ????43 |
| ??DcGTX 1 | ????50 | ????171 | ????45 |
| ??DcGTX 2 | ????380 | ????68 | ????18 |
| ??DcGTX 3 | ????380 | ????68 | ????18 |
| ??dcGTX 4 | ????950 | ????171 | ????45 |
The active preliminary study of PSP toxin is shown that the PSP toxin has local anesthetic action to nervous system, and Adams etc. have applied for multinomial local anaesthesia patent.In existing patent documentation CN1192903, disclosed the PSP toxin as drug-breaking medicine.Mice treatment detoxification evidence PSP toxin by the perpendicular tail test of mice, mouse jump reaction test, the body weight loss of weight alternate test that relies on the morphine rat, the test of monkey addiction, dependence morphine, cocaine in the document does not have drug dependence; And the PSP toxin is used to give up the drug dependence of addiction medicine product such as opium, heroin, morphine, Pethidine, and the clinical good result that presents, in the dosage range of strict regulations, toxicity, side effect is little.In addition, also have report to think that the PSP toxin has hypotensive effect to cardiovascular, and muscle is had relexation.But yet there are no so far the report of PSP toxin as analgesics.
Pain is indication phenomenon reality or potential injury or tissue injury that causes as inflammation, ischemia, machinery and other stimulation.Pain and anthropogeny are almost taken place simultaneously, fail the mystery explained fully but be still so far.In all age, people have used many methods that ease the pain, and stroke, push, rub elementary primary methods such as wiping from application, to using hot spring, hot compress, arrive and use natural herbal external application analgesia.At present treatment of pain is comprised and use the block nerves conduction, and influence the local anesthetic and the alleviating pain of pain and may attach the active analgesic of interference inflammation chemical messenger.But undeniable some intractable pains that still have as carcinomas pain, neuralgia etc., even take medicine by operation method or internal medicine, can not take effect fully.As cancer pain at late stage, often adopt opium kind analgesics to ease pain, but be easy to be addicted, produce drug resistance, and follow constipation, feel sick, side effect such as vomiting.Though the report that local anesthetic is used for easing the pain is also arranged, to lack because of its acting duration, frequent administration of needs and toxic and side effects are big etc., and effect is not good usually.
(3) technical scheme:
The present invention is intended to overcome long, deficiency such as habit-forming easily of existing analgesic (as Pethidine) persistent period, seeks a kind of curative effect height, the long analgesic of acting duration.The present invention intends exploitation and contains the associating of PSP toxin and analgesic, or with the compositions of the material formation that pharmaceutically allows.Find that after deliberation the PSP toxin can use separately, and analgesic activity intensity is big, acting duration is long; Also can unite use, can prolong the analgesic activity persistent period, reduce toxic and side effects by reducing the drug use amount with analgesic.
It is to have selected following five classes in this PSP toxin of kind more than 20, to have included: (one) carbamate toxoid (carbamatetoxins) that paralytic shellfish poison (PSP) toxin of the present invention's research is treated the application of controlling the antalgesic medicine as preparation; (2) N-sulphonyl carbamyl toxoid (N-sulfocarbamoyl toxins); (3) deamination formoxyl toxoid (decarbamoyl toxins); (4) deoxidation deamination formoxyl toxoid (deoxydecarbamoyltoxins); (5) the N-hydroxy derivatives of carbamates (N-hydroxycarbamoyl derivatives) is wherein selected a class or a few class.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, described (one) carbamate toxoid (carbamate toxins), it comprises: (1) saxitoxin (STX), (2) new saxitoxin (neoSTX), (3) gonyatoxin 1 (GTX
1), (4) gonyatoxin 2 (GTX
2), (5) gonyatoxin 3 (GTX
3), (6) gonyatoxin 4 (GTX
4); Wherein select one or more;
Described (two) N-sulphonyl carbamyl toxoid (N-sulfocarbamoyl toxins), it comprises: (1) gonyatoxin 5 (GTX
5), (2) gonyatoxin 6 (GTX
6), (3) 21-N-sulphonyl carbamyl toxin 1 (C
1), (4) 21-N-sulphonyl carbamyl toxin 2 (C
2), (5) 21-N-sulphonyl carbamyl toxin 3 (C
3), (6) 21-N-sulphonyl carbamyl toxin 4 (C
4);
Described (three) deamination formoxyl toxoid (decarbamoyl toxins); it comprises: (1) deamination formoxyl saxitoxin (dcSTX); (2) DcneoSTX (dcneoSTX), (3) dcGTX1 Decarbamoylgonyautoxin 1. (dcGTX
1), (4) dcGTX2 Decarbamoylgonyautoxin 2. (dcGTX
2), (5) dcGTX3 (dcGTX
3), (6) dcGTX4 Decarbamoylgonyautoxin 4. (dcGTX
4);
Described (four) deoxidation deamination formoxyl toxoid (deoxydecarbamoyl toxins) comprising: (1) deoxidation deamination formoxyl saxitoxin (doSTX), (2) deoxidation dcGTX2 Decarbamoylgonyautoxin 2. (doGTX
2), (3) deoxidation dcGTX3 (doGTX
3);
The N-hydroxy derivatives (N-hydroxycarbamoyl derivatives) of described (five) carbamates, it comprises: (1) 21-N-STX-OL (hySTX), the new saxitoxin of (2) 21-N-hydroxyl (hyneoSTX).
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, described (one) carbamate toxoid (carbamate toxins), (1) saxitoxin (STX) wherein, the range of application of (2) new saxitoxin (neoSTX) exists: 0.01-10 μ g/kg; (3) gonyatoxin 1 (GTX
1), (6) gonyatoxin 4 (GTX
4) range of application exist: 0.01-15 μ g/kg; (4) gonyatoxin 2 (GTX
2), (5) gonyatoxin 3 (GTX
3) range of application exist: 0.01-20 μ g/kg.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, described (two) N-sulphonyl carbamyl toxoid (N-sulfocarbamoyl toxins), wherein
(1) gonyatoxin 5 (GTX
5),
(2) gonyatoxin 6 (GTX
6),
(3) 21-N-sulphonyl carbamyl toxin 1 (C
1),
(4) 21-N-sulphonyl carbamyl toxin 2 (C
2) range of application all exist: 0.1-100.0 μ g/kg.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, described (three) deamination formoxyl toxoid (decarbamoyl toxins), wherein
(1) deamination formoxyl saxitoxin (dcSTX),
(2) DcneoSTX (dcneoSTX),
(3) dcGTX1 Decarbamoylgonyautoxin 1. (dcGTX
1),
(6) dcGTX4 Decarbamoylgonyautoxin 4. (dcGTX
4) range of application all exist: 0.02-20 μ g/kg;
(4) dcGTX2 Decarbamoylgonyautoxin 2. (dcGTX
2),
(5) dcGTX3 (dcGTX
3) range of application all exist: 0.05-50 μ g/kg.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine, described (one) carbamate toxoid (carbamatetoxins) as preparation; (2) N-sulphonyl carbamyl toxoid (N-sulfocarbamoyltoxins); (3) deamination formoxyl toxoid (decarbamoyl toxins); (4) deoxidation deamination formoxyl toxoid (deoxydecarbamoyl toxins); (5) the N-hydroxy derivatives of carbamates (N-hydroxycarbamoyl derivatives) is wherein selected a class or a few class, or/and wherein one or more can also with narcosis analgesic thing use in conjunction;
Wherein the narcosis analgesic thing has: morphine, codeine, Pethidine, anadol, fentanyl, methadone, pentazocine, dihydroetorphine, tramadol, bucinnazine, tetrahydropalmatine, rotundine, naloxone, naltrexone, choose wherein one or more and above-mentioned (one)---a class or a few class in (five) toxoid, form compositions or/and one or more mix mutually.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, described (one) carbamate toxoid (carbamate toxins), wherein preferred (4) gonyatoxin 2 (GTX
2), (5) gonyatoxin 3 (GTX
3) with the ratio that cooperates of analgesic use in conjunction be: (weight ratio)
The PSP toxin name of an article: analgesic medicine name:
Gonyatoxin 2 (GTX
2)
Or gonyatoxin 3 (GTX
3): 1.0-7.5 μ g/kg, Pethidine: 1-20mg/kg.
Paralytic shellfish poison of the present invention (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, can mix forming compositions by acceptable carrier, excipient, adjuvant, diluent, slow releasing agent, controlled release agent on effective dose and the materia medica, be used to prepare analgesics.
This paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, can make various dosage forms according to prior art, as make injection, comprise subcutaneous, muscle, intravenous injection and oral agents, comprise the sublingual administration agent and, comprise aerosol and microcapsule etc. by the respiratory tract inhalant.But the effective dose that oral effective dose will be higher than injection and suck, the 5-100 that the former is about the latter doubly, so optimizing injection.The present invention research is also found can be by the mode that this PSP toxin and analgesic are dissolved in mode together or separate.
PSP toxin of the present invention is used proof as treatment antalgesic medicine through clinical trial: really can inhibition of pain, and action intensity is big, and acting duration is long.This PSP toxin and analgesic use in conjunction can prolong the analgesic activity persistent period, reduce the consumption of analgesic, thereby reduce the toxic and side effects of analgesic.
(4) embodiment and accompanying drawing thereof:
The embodiments of the invention accompanying drawings is as follows.
Protection scope of the present invention not only is confined to following embodiment.
Fig. 1 is: the PSP toxin suppresses the reversibility of mouse movement teleneuron sodium stream;
Fig. 2 is: the PSP toxin suppresses the reversibility of NG108-15 cellular sodium stream;
Fig. 3 is: the threshold of pain with medicine after the variation of time
Present embodiment is measured the PSP toxin to the effect of sodium stream and the potassium stream of mouse movement nerve endings and NG108-15 cell.
(1) materials and methods
1.1 the PSP toxin is to the influence of mouse movement teleneuron sodium stream
Sample solution preparation: the sample that the PSP toxin is made into 230nmol/L with the hydrochloric acid solution of 0.1mol/L.
1.1.1 the preparation of sample and maintenance
Experiment is carried out on the stripped intercostal nerves breast triangular muscle specimen of adult mice (male and female are all used for kunming mice, body weight 18-20g).Specimen press McArdle etc. (Mcardle, J.J.et al., J.Neurosci.Methods., 1981,4:109-115.) method preparation is by ventilation (95%O
2And 5%CO
3Mist) improvement Krebs standard solution continous perfusion, perfusion rate is 2-4ml/min.Standard solution consists of (mmol/L): NaCl 138; KCl5; CaCl
22; MgCl
21; NaH
2 PO
41; NaHCO
312; Glucose 10, pH 7.2-7.4.For preventing muscle contraction, in whole experiment, perfusate remains the tubocurare of finite concentration (50-100 μ mol/L).In this concentration range, tubocurare does not all produce visible influences to sodium, potassium stream.Experiment is carried out under room temperature (18-25 ℃) condition.
1.1.2 bringing out and record of tip membrane current
Stimulate intercostal nerves with a homemade capillary glass tube adsorption electrode, parameter is the wide 100 μ s supraliminal stimulus of ripple, and frequency is generally 0.1Hz (unless specializing).The glass microelectrode that recording electrode system draws with the GG-17 horminess glass tube, in charge 1mol/L NaCl solution, resistance is between 5-10M Ω, guide with the Ag/AgCl silk, reference electrode is for embedding the Ag/AgCl sheet in the specimen groove, by means of water immersion objective (* 40, Zeiss), at the Zeiss microscopically that amplifies 400 times, recording electrode is inserted to gap under neural all films of the nearly soleplate of the thin nerve tract in top layer.Signal under the neural all films that record (perineurial signal) is through microelectrode amplifier (MEZ-8201 Nikon-kohden, Tokyo), one the tunnel is shown in two-wire memory oscilloscope (Tektronix 5113A), (Digidata 1200 through the AD/DA change-over panel on another road, Axon) by the computer sampling storage signal, use Axoscope, CoralDraw 8.0 softwares are drawn.
1.2PSP toxin is to the effect of the sodium stream and the potassium stream of NG108-15 cell
1.2.1 the cultivation of NG108-15 cell
The NG108-15 cell is so kind as to give by Japan's Physiologic Studies.Keeping cell is the DMEM solution that contains 10% calf serum, 100 μ mol/Lhypoxanthine, 1 μ mol/Laminopterin, 16 μ mol/Lthymidine in the standard culture liquid of merisis state, keeps cell in the merisis state.With cell inoculation in Tissue Culture Flask, at 37 ℃ 5%CO
2, cultivate in the 95% air cell culture incubator, changed one time culture fluid every 2-3 days.After treating that cell in the culture bottle covers with, just can be used for going down to posterity or breaking up.On the basis of standard culture liquid, the concentration of calf serum is reduced to 5%, and increase 1mmol/L dEcAMP (N
6O
2-dibutyryl adenosine 3 ', 5-cyclic monophosphate) in culture fluid, impel cell differentiation, the cell that will be used to break up is by 1-4 * 10
4The density of cell/culture dish is inoculated in the 35mm plastic culture dish, at 37 ℃ 5%CO
2, cultivate in the 95% air cell culture incubator, changed one time culture fluid every 2-3 days.What be generally used for testing is the cell that has broken up 5-9 days.
1.2.2 full cell record
Experiment is carried out under full cell voltage pincers.Experimental system is by inverted phase contrast microscope, narishige, perfusion system, computer, A/D, D/A data input/output (Scientific Solutions, Inc.), patch clamp amplifier (CBZ2300 Nikon-kohden, composition such as Tokyo), all experiments are carried out under pClamp 5.51 controls.
Perfusion system is homemade Y type perfusion device, a plastic capillary that port is the about 100 μ m of diameter of Y-piece, and two ports in addition of Y-piece, in the perfusion solution that one is immersed in the beaker, the negative pressure bottle of another UNICOM's one valve control.Open the solution in the replaceable Y-piece of valve, perfusate is gushed out from capillary tube by the potential difference of solution, and flow velocity is about 0.2ml/min.The external diameter that the glass microelectrode that is used to test is produced by Shanghai physiology is that the material glass microelectrode blank of 1.5mm forms through twice drawing of NARISHIGE PP-83 electrode drawing device, is in the electrode behind the liquid with high potassium, and electrode impedance is 2-5M Ω.
Experiment is carried out under room temperature (18-25 ℃), track is fixed on the platform of inverted phase contrast microscope, under the extracellular fluid perfusion of standard, make filling have the electrode of liquid in the electrode to contact with cell surface, gently inhale, make electrode and cell membrane form the high impedance sealing-in, inhale again, cell membrane is inhaled broken, form full cell record pattern.Observe sodium stream and potassium stream, (mmol/LKCl 5 to use the interior liquid of high potassium electrode; NaCl 145; MgCl
20.8; CaCl
21.8; Glucose 30; HEPES 20).Experiment is under pClamp 5.51 programme-control, by computer control patch clamp amplifier cell is clamped on the required clamping voltage (holding potential) of experiment, give cell with required test voltage (testing potential), can observe the cell transmembrane electric current that test voltage is brought out.
(2) result
2.1 reversibility ground suppresses the sodium stream of mouse movement teleneuron
After in Krebs solution, recording two negative spikes and waiting signal stabilization, with thick toxin of PSP that contains 50 μ l/10ml Krebs liquid (concentration is 1.18nmol/L STXeq) and STX normaltoxin (concentration is 3.94nmol/L) difference perfusion specimen, the waveform of all finding record has significant change, first negative peak obviously reduces, and shows that sodium current is obviously suppressed.The effect of two samples is reversible, and with the solution flushing that does not contain sample, inhibition can partly be removed, and signal recovers gradually to some extent.
With STX normaltoxin (concentration is 3.94nmol/L) perfusion specimen, the waveform of record has significant change in 2 minutes, and first negative peak obviously reduces, and shows that sodium current is obviously suppressed (Fig. 1 Sample A 2min), second negative peak also obviously reduces, and shows that potassium current is also obviously suppressed.With Krebs standard solution flushing 15 minutes, inhibition can partly be removed immediately, and signal recovers (Fig. 1 Wash 15min) gradually to some extent.With the thick toxin of PSP (concentration is 1.18nmol/L STXeq) perfusion specimen, the waveform of record has significant change in 2 minutes, first negative peak obviously reduces, show that sodium current is obviously suppressed (Fig. 1 Sample B 2min), second negative peak also obviously reduces, and shows that potassium current is also obviously suppressed; In 6 minutes the record first negative peak very low, show sodium current major part be suppressed (Fig. 1 Sample B 6min); Suppressed fully gradually in 7 minutes (Fig. 1 Sample B 7min), 8 minutes (Fig. 1 Sample B 8min) sodium currents.Corresponding to the inhibition that 2,6,7,8 minutes sodium currents are subjected to, the inhibition amplitude that potassium current is subjected to is also increasing, until being suppressed fully.
2.2 reversibility ground suppresses the sodium stream of NG108-15 cell
After obtaining full cell record configuration, with the outer liquid continous perfusion cell of standard cell lines, then with thick toxin of PSP (concentration is 4.70nmol/L STXeq) that contains 200 μ l/10ml extracellular fluids and STX normaltoxin (concentration is 15.74nmol/L) difference perfusion cell, the sodium current of all observing cell obviously is suppressed, and the variation of potassium current is little.This inhibitory action is reversible, can observe sodium current with the solution perfusion that does not contain sample and recover to some extent.Fig. 2 has write down the routine result with STX standard solution perfusion cell.With STX normaltoxin (concentration is 15.74nmol/L) perfusion cell, the sodium current of observing cell obviously is suppressed, and the variation of potassium current is little.Can observe sodium current with the solution perfusion that does not contain sample recovers to some extent.
(3) conclusion
The PSP toxin can suppress to reversibility the sodium stream of mouse movement teleneuron and NG108-15 cell.
Present embodiment adopts mouse writhing to test the analgesic effect of measuring the PSP toxin.
(1). material and method
The aspirin enteric coatel tablets, the 0.3g/ sheet, lot number 9804292, Huaiyin, Jiangsu pharmaceutical factory produces.Kunming mouse, the cleaning level is provided by Qingdao City's laboratory animal and zoopery center, and animal produces the quality certification: No. 970209, Shandong kinoplaszm word.
The sample solution preparation: it is an amount of to get PSP toxin sample (26 μ g STX equivalent/ml), be made into 3.2 μ g STX equivalent/ml with distilled water, 1.6 μ g STX equivalent/ml, 0.4 μ g STX equivalent/ml, 0.2 the solution of μ g STX equivalent/ml and 0.1 μ g STX equivalent/ml is respectively applied for PSP toxin 80 μ g STX equivalent/kg group, 40 μ g STX equivalent/kg group, 10 μ g STX equivalent/kg group, 5 μ g STX equivalent/kg group and 2.5 μ g STX equivalent/kg group.
The solvent solutions preparation: get solvent 1ml, adding distil water 64ml is used for negative control group.
Aspirin solution preparation: get the aspirin enteric coatel tablets and be made into the solution of 8mg/ml by aspirin content with distilled water in right amount, be used for positive controls.
Get 870 of 18-22g mices, be divided into totally 8 groups of PSP toxin 80 μ g STX equivalent/kg group, 40 μ gSTX equivalent/kg group, 10 μ g STX equivalent/kg group, 5 μ g STX equivalent/kg group and 2.5 μ gSTX equivalent/kg group, positive controls (aspirin 0.2g/kg), negative control group (solvent), blank groups (normal saline) at random.Each component of medicine gives mouse stomach with PSP toxin 80 μ g STXequivalent/kg group, 40 μ g STX equivalent/kg group, 10 μ g STX equivalent/kg group, 5 μ g STXequivalent/kg group and 2.5 μ g STX equivalent/kg group respectively, positive controls gives mouse stomach with aspirin by 200mg/kg, negative control group with joining solvent solutions give mouse stomach by 25ml/kg, the blank group gives mouse stomach with normal saline by 25ml/kg, and 30 minutes lumbar injection 0.6% glacial acetic acid 0.2ml/ only after administration.Observe in 15 minutes animal and turn round the body number of times.
(2). result: see Table 3.Table 3 PSP toxin is to the analgesic activity (writhing method) of mice
Number of animals is turned round the body number of times
Group P
(only) (% ± SD)
(3). conclusion
According to the statistical analysis of experimental result, the PSP toxin can significantly reduce the mice pain reaction number of times that is caused by acetic acid when according to dosage 5 μ g STX equivalent/kg give mouse stomach, has significant analgesia role.
Embodiment 3
Present embodiment adopts the mice hot plate test to measure analgesic effect.
(1). method
Screening: get some of 19-21g body weight female mices, be put in the beaker that is heated to 55 ℃ in advance, be fed into the response time (incubation period) that metapedes occurs licking with the stopwatch record from mice, with this as threshold of pain index, to react too responsive, blunt or happiness jumping person rejects, the response latency mice in the 10-30s scope be used for experiment.
Grouping: will divide equally eight groups at random by body weight: 0.1mol/L acetic acid solvent negative control group, the GTX that determines according to the preliminary experiment result through screening 72 of qualified mices
2,3High dose (7.49 μ g STX equivalent/kg), middle dosage (5.99 μ g STX equivalent/kg) and low dosage (4.79 μ g STX equivalent/kg) three reagent groups, pethidine hydrochloride high dose (10mg/kg) and low dosage (5mg/kg) two positive controls, and low dosage GTX
2,3(4.79 μ g STX equivalent/kg)+low dosage pethidine hydrochloride (5mg/kg) (drug combination low dose group), low dosage GTX
2,3(4.79 μ g STX equivalent/kg)+high dose pethidine hydrochloride (10mg/kg) (drug combination high dose group) two drug combination groups.
Each is organized equal i.m and gives relative medicine, according to said method different time measured reaction incubation period before medicine, behind the medicine.
Statistical method: check self relatively and between each group comparing of carrying out the administration front and back with t.
(2). result: GTX
2,3Certain analgesic activity is arranged, and with the pethidine hydrochloride use in conjunction, have the analgesia synergism.See Fig. 3 and table 4.
Table 4 GTX
2,3Analgesic activity to mice
Before the medicine behind the pain threshold medicine during threshold of pain, (s) group, (s) 5 ' 10 ' 15 ' 20 ' 30 ' 40 ' 60 ' 90 ' 120 ' 150 ' 180 ' 210 ' 240 ' negative control group mol/L acetic acid
0.1???????20.30?21.89?????22.67?????20.89?????20.56?????18.78?????22.11?????21.11??????21.22?????23.44?????21.56?????22.32?????21.00???21.22
± 4.95 ± 6.41 ± 6.73 ± 7.74 ± 7.54 ± 5.19 ± 7.79 ± 9.17 ± 6.32 ± 6.17 ± 3.54 ± 5.98 ± 5.24 ± 5.49 Pethidine mg/kg
10???23.22??53.22?????48.11?????45.44?????42.00?????39.44?????27.30???????27.40?????25.20
±3.63?±8.29
**±12.63
**±13.60
**±13.57
**±16.50
**±14.9??????±13.80???±4.89
5????22.00??25.00?????27.11?????24.33?????25.33?????25.78?????25.11???????25.22?????22.44
±5.05?±6.80???±9.29????±7.21????±6.65????±10.44???±5.25??????±3.49????±5.81GTX
2，3μg?STX?equivalent/kg
7.49?20.56??41.56?????55.44?????57.67????59.56?????59.67????＞60
**??????59.11?????55.67??????47.11?????43.67?????35.78???22.00
±3.39?±13.99
**±9.21
**±4.95
**±1.33
**?±1.00
**?????????????±2.67
**??±11.25
**±16.37
**±16.36
**±15.93
**±2.52
*
5.99?21.78??39.33?????50.78?????55.22?????57.00?????52.00?????47.56???????35.22?????36.56?????35.56?????25.33
±4.79?±14.59
**±11.74
**±6.53
**?±7.31
**?±10.34
**±12.77
**??±13.83
**±15.07
**±14.22
*?±6.30
4.79?21.56??25.22?????31.67?????26.78?????30.11?????31.89?????30.89???????24.22?????23.67
± 6.29 ± 6.50 ± 10.51
*± 8.79 ± 9.35
*± 8.02
*± 10.97 ± 4.92 ± 4.69 drug combination high doses 23.44 43.67 49.00 55.33 56.67 58.00 55.11 44.89 39.22 39.11 35.78
± 3.78 ± 16.03
*± 13.51
*± 9.59
*± 6.73
*± 3.50
*± 10.02
*± 12.81
*± 12.33
*± 13.04
*± 14.45
*Low dosage 20.00 31.78 35.78 38.56 35.22 33.67 31.33 23.78 23.00
±5.24?±10.86
*?±12.01
**±10.99
**±12.16
**±9.73
**?±9.67
*????±5.91????±4.61
* * and negative control group be p＜0.05 p＜0.01 relatively
From onset time, the not obvious and GTX of demineralizing acid Pethidine low dose group effect
2,3Outside the low dose group 10min onset, other all 5min onset behind medicine of each administration group, the action intensity of this moment by by force to a little less than be followed successively by: the highest during the threshold of pain of pethidine hydrochloride high dose (10mg/kg), promptly the response latency the longest, be 53.22 seconds; Drug combination high dose, GTX
2,3High dose, middle dosage, drug combination low dosage were respectively during the threshold of pain 43.67 seconds, 41.56 seconds, 39.33 seconds and 31.78 seconds, more all had significantly with negative control group or extremely remarkable meaning (p＜0.05 orp＜0.01).
From the effect peak time, the pethidine hydrochloride high dose group, behind the medicine 5min time effect the strongest, other each medication group 15-40min behind medicine does not wait.During the threshold of pain peaking by by force to a little less than be followed successively by: GTX
2,3High dose (60min), the drug combination positive (58min), GTX2, dosage (57min), pethidine hydrochloride high dose (53.22min), drug combination low dosage (38.56min) and GTX in 3
2,3Low dosage (31.89min) compares with the negative control group peak, and extremely significantly meaning (P＜0.01) is all arranged.
Hold time from effect: pethidine hydrochloride positive control dosage group 10min behind medicine promptly begins to recover, and recovers (self contrast) during 40min fully; GTX
2,3High, middle dosage group and drug combination high dose group animal majority 2-3h behind medicine recovers, and minority can extend to 4-5.5h, GTX
2,3Behind medicine, recover fully in the 60min during threshold of pain of low dosage and drug combination low dose group animal.
In a word: no matter be from onset time, action intensity, or effect hold time, when singly using GTX
2,3Or when pethidine hydrochloride or drug combination, all the show dose dependency in advance, strengthen and prolong.
It should be noted that behind drug combination relatively action intensity increases in advance analgesic activity onset time with dosage with the folk prescription medication, acting duration prolongs.And the reduction of folk prescription dose means that toxicity reduces.
Embodiment 4
The analgesic effect of various PSP toxin is listed in table 5.The analgesic effect of the various PSP toxin of table 5
+, ++, +++represent respectively analgesic effect weak, in, strong.
| Numbering | The toxin title | Dosage μ g/kg | Analgesic effect | Dosage μ g/kg | Analgesic effect | Dosage μ g/kg | Analgesic effect | Dosage μ g/kg | Analgesic effect | Dosage μ g/kg | Analgesic effect |
| ??1 | ??STX | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????10 | ??+++ |
| ??2 | ??neoSTX | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????10 | ??+++ |
| ??3 | ??GTX1 | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????15 | ??+++ |
| ??4 | ??GTX4 | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????15 | ??+++ |
| ??5 | ??GTX2 | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????20 | ??+++ |
| ??6 | ??GTX3 | ??0.01 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????2 | ????++ | ????20 | ??+++ |
| ??7 | ??GTX5 | ??0.1 | ????+ | ????1 | ????+ | ????10 | ????+ | ????50 | ????++ | ????100 | ??+++ |
| ??8 | ??GTX6 | ??0.1 | ????+ | ????1 | ????+ | ????10 | ????+ | ????50 | ????++ | ????100 | ??+++ |
| ??9 | ??C1 | ??0.1 | ????+ | ????1 | ????+ | ????10 | ????+ | ????50 | ????++ | ????100 | ??+++ |
| ??10 | ??C2 | ??0.1 | ????+ | ????1 | ????+ | ????10 | ????+ | ????50 | ????++ | ????100 | ??+++ |
| ??11 | ??dcSTX | ??0.02 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????1 | ????++ | ????20 | ??+++ |
| ??12 | ??dcneoSTX | ??0.02 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????1 | ????++ | ????20 | ??+++ |
| ??13 | ??dcGTX1 | ??0.02 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????1 | ????++ | ????20 | ??+++ |
| ??14 | ??dcGTX4 | ??0.02 | ????+ | ????0.1 | ????+ | ????0.5 | ????+ | ????1 | ????++ | ????20 | ??+++ |
| ??15 | ??dcGTX2 | ??0.05 | ????+ | ????0.1 | ????+ | ????1 | ????+ | ????10 | ????++ | ????50 | ??+++ |
| ??16 | ??dcGTX3 | ??0.05 | ????+ | ????0.1 | ????+ | ????1 | ????+ | ????10 | ????++ | ????50 | ??+++ |
Claims (8)
1, a kind of paralytic shellfish poison (PSP) toxin is treated the application of controlling the antalgesic medicine as preparation, and it is characterized in that: this PSP toxin includes: (one) carbamate toxoid; (2) N-sulphonyl carbamyl toxoid; (3) deamination formoxyl toxoid; (4) deoxidation deamination formoxyl toxoid; (5) the N-hydroxy derivatives of carbamates; Wherein select a class or a few class.
2, treat the application of controlling the antalgesic medicine as preparation according to the described paralytic shellfish poison of claim 1 (PSP) toxin, it is characterized in that: described (one) carbamate toxoid, it comprises: (1) saxitoxin (STX), (2) new saxitoxin (neoSTX), (3) gonyatoxin 1 (GTX
1), (4) gonyatoxin 2 (GTX
2), (5) gonyatoxin 3 (GTX
3), (6) gonyatoxin 4 (GTX
4); Wherein select one or more;
Described (two) N-sulphonyl carbamyl toxoid, it comprises: (1) gonyatoxin 5 (GTX
5), (2) gonyatoxin 6 (GTX
6), (3) 21-N-sulphonyl carbamyl toxin 1 (C
1), (4) 21-N-sulphonyl carbamyl toxin 2 (C
2), (5) 21-N-sulphonyl carbamyl toxin 3 (C
3), (6) 21-N-sulphonyl carbamyl toxin 4 (C
4); Wherein select one or more;
Described (three) deamination formoxyl toxoid, it comprises: (1) deamination formoxyl saxitoxin (dcSTX), (2) DcneoSTX (dcneoSTX), (3) dcGTX1 Decarbamoylgonyautoxin 1. (dcGTX
1), (4) dcGTX2 Decarbamoylgonyautoxin 2. (dcGTX
2), (5) dcGTX3 (dcGTX
3), (6) dcGTX4 Decarbamoylgonyautoxin 4. (dcGTX
4); Wherein select one or more;
Described (four) deoxidation deamination formoxyl toxoid, it comprises: (1) deoxidation deamination formoxyl saxitoxin (doSTX), (2) deoxidation dcGTX2 Decarbamoylgonyautoxin 2. (doGTX
2), (3) deoxidation dcGTX3 (doGTX
3); Wherein select one or more;
The N-hydroxy derivatives of described (five) carbamates, it comprises: (1) 21-N-STX-OL (hySTX), the new saxitoxin of (2) 21-N-hydroxyl (hyneoSTX); Wherein select one or more.
3, treat the application of controlling the antalgesic medicine according to claim 1 or 2 described paralytic shellfish poison (PSP) toxin as preparation, it is characterized in that: described (one) carbamate toxoid, wherein
(1) saxitoxin (STX), the range of application of (2) new saxitoxin (neoSTX) exists: 0.01-10 μ g/kg;
(3) gonyatoxin 1 (GTX
1), (6) gonyatoxin 4 (GTX
4) range of application exist: 0.01-15 μ g/kg;
(4) gonyatoxin 2 (GTX
2), (5) gonyatoxin 3 (GTX
3) range of application exist: 0.01-20 μ g/kg.
4, treat the application of controlling the antalgesic medicine according to claim 1 or 2 described paralytic shellfish poison (PSP) toxin as preparation, it is characterized in that: described (two) N-sulphonyl carbamyl toxoid, wherein
(1) gonyatoxin 5 (GTX
5),
(2) gonyatoxin 6 (GTX
6),
(3) 21-N-sulphonyl carbamyl toxin 1 (C
1),
(4) 21-N-sulphonyl carbamyl toxin 2 (C
2) range of application all exist: 0.1-100.0 μ g/kg.
5, treat the application of controlling the antalgesic medicine according to claim 1 or 2 described paralytic shellfish poison (PSP) toxin as preparation, it is characterized in that: described (three) deamination formoxyl toxoid, wherein
(1) deamination formoxyl saxitoxin (dcSTX),
(2) DcneoSTX (dcneoSTX),
(3) dcGTX1 Decarbamoylgonyautoxin 1. (dcGTX
1),
(6) dcGTX4 Decarbamoylgonyautoxin 4. (dcGTX
4) range of application all exist: 0.02-20 μ g/kg;
(4) dcGTX2 Decarbamoylgonyautoxin 2. (deGTX
2),
(5) dcGTX3 (dcGTX
3) range of application all exist: 0.05-50 μ g/kg,
6, treat the application of controlling the antalgesic medicine as preparation according to claim 1 or 2 described paralytic shellfish poison (PSP) toxin, it is characterized in that: the N-hydroxy derivatives of described (one) carbamate toxoid, (two) N-sulphonyl carbamyl toxoid, (three) deamination formoxyl toxoid, (four) deoxidation deamination formoxyl toxoid, (five) carbamates, choose wherein a class or a few class, or/and wherein one or more and narcosis analgesic thing use in conjunction;
Wherein the narcosis analgesic thing has: morphine, codeine, Pethidine, anadol, fentanyl, methadone, pentazocine, dihydroetorphine, tramadol, bucinnazine, tetrahydropalmatine, rotundine, naloxone, naltrexone, choose wherein one or more and above-mentioned (one)---a class or a few class in (five) toxoid, form compositions or/and wherein one or more mix mutually.
7, treat the application of controlling the antalgesic medicine according to the described paralytic shellfish poison of claim 6 (PSP) toxin as preparation, it is characterized in that: described (one) carbamate toxoid, wherein preferred
(4) gonyatoxin 2 (GTX
2),
(5) gonyatoxin 3 (GTX
3) with the ratio that cooperates of analgesic use in conjunction be: (weight ratio)
The PSP toxin name of an article: analgesic medicine name:
Gonyatoxin 2 (GTX
2)
Or gonyatoxin 3 (GTX
3): 1.0-7.5 μ g/kg, Pethidine: 1-20mg is with kg.
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|---|---|---|---|
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1443932A4 (en) * | 2001-11-15 | 2007-11-28 | Micro Algae Corp | Pharmaceutical compositions containing 3,4-propinoperhydropurines and uses thereof for blocking neuronal transmission |
| US8377951B2 (en) | 2004-05-07 | 2013-02-19 | Phytotox Limited | Transdermal administration of phycotoxins |
| US20160115173A1 (en) * | 2009-05-07 | 2016-04-28 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for studying, imaging, and treating pain |
| US10106549B2 (en) | 2014-04-09 | 2018-10-23 | Siteone Therapeutics, Inc. | 10′,11′-modified saxitoxins useful for the treatment of pain |
| US10112953B2 (en) | 2015-09-30 | 2018-10-30 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
| US11236097B2 (en) | 2017-03-29 | 2022-02-01 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
| US11279706B2 (en) | 2017-03-29 | 2022-03-22 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
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2002
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1443932A4 (en) * | 2001-11-15 | 2007-11-28 | Micro Algae Corp | Pharmaceutical compositions containing 3,4-propinoperhydropurines and uses thereof for blocking neuronal transmission |
| US8871763B2 (en) | 2001-11-15 | 2014-10-28 | Phytotox, Ltd. | Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine, for cosmetology, wound healing, focal dystonia and muscular spasm- related clinical pathologies |
| US8889681B2 (en) | 2001-11-15 | 2014-11-18 | Korea Research Institute Of Chemical Technology | Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine, for cosmetology, wound healing, focal dystonia and muscular spasm-related clinical pathologies |
| US9301958B2 (en) | 2001-11-15 | 2016-04-05 | Phytotox Limited | Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine |
| US8377951B2 (en) | 2004-05-07 | 2013-02-19 | Phytotox Limited | Transdermal administration of phycotoxins |
| US20160115173A1 (en) * | 2009-05-07 | 2016-04-28 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for studying, imaging, and treating pain |
| US10513525B2 (en) * | 2009-05-07 | 2019-12-24 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for studying, imaging, and treating pain |
| US10106549B2 (en) | 2014-04-09 | 2018-10-23 | Siteone Therapeutics, Inc. | 10′,11′-modified saxitoxins useful for the treatment of pain |
| US10112953B2 (en) | 2015-09-30 | 2018-10-30 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
| US11236097B2 (en) | 2017-03-29 | 2022-02-01 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
| US11279706B2 (en) | 2017-03-29 | 2022-03-22 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
| US11834456B2 (en) | 2017-03-29 | 2023-12-05 | Siteone Therapeutics, Inc. | 11,13-modified saxitoxins for the treatment of pain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1194693C (en) | 2005-03-30 |
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