CN102344950B - A kind of method of the acetylize metabolite for detecting SSAT substrate - Google Patents

A kind of method of the acetylize metabolite for detecting SSAT substrate Download PDF

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CN102344950B
CN102344950B CN201110145069.XA CN201110145069A CN102344950B CN 102344950 B CN102344950 B CN 102344950B CN 201110145069 A CN201110145069 A CN 201110145069A CN 102344950 B CN102344950 B CN 102344950B
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ssat
acetylize
metabolite
detecting
inhibitor
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CN102344950A (en
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郑启明
阿曼得·拉希德
李铁军
周辉
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Biomark(China) International Ltd
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Abstract

The present invention relates to medical art, be specifically related to a kind of spermidine/spermine N 1the method of-acetyltransferase activity, namely by detecting the quantitative technique of the acetylize metabolite analysis SSAT activity of SSAT substrate.The present invention finds mainly or only to regulate the acetylizad specific substrate of N-to be levodopa by SSAT in SSAT path; and the part metabolism of this substrate can produce acetylize metabolite N-acetyl base-L-3,4 dihydroxyphenylalanine by the induction of SSAT; SSAT raises and is proportionate with cancer; therefore; the cancer markers that levodopa can regulate as SSAT, for finding the morning of tumour, early the aspect such as diagnosis and early treatment provide a kind of new detection method.The inventive method is safe and effective, and practicality is comparatively strong, and its working method is convenient and swift, easy to use, and positive rate is high, Be very effective, can be used for preventing, diagnosis, detect, protection, treatment and study of various tumour supplementary means.

Description

A kind of method of the acetylize metabolite for detecting SSAT substrate
Technical field
The present invention relates to medical art; specifically relate to a kind of medical detection technique; more particularly relate to a kind of spermidine/spermine N1-Transacetylase (be called for short: the SSAT) detection method of the acetylize metabolite of the SSAT substrate that activity methods is relevant; namely the acetylize metabolite by detecting SSAT substrate analyzes SSAT activity; wherein: SSAT substrate is levodopa, the part metabolism of levodopa can produce acetylize metabolite N-acetyl base-L-3,4 dihydroxyphenylalanine by the induction of SSAT.
Background technology
(1) research overview of levodopa
1, summarize
L-Dopa/L-β-(3,4-dihydroxy-benzene) L-Ala/L-3-(3,4-dihydroxy-benzene) L-Ala L-(3,4-dihydroxy-benzene) L-Ala (Levodopa), (be called for short: L-Dopa also known as L-3,4 dihydroxyphenylalanine, L-DOPA), being the hydroxylate of tyrosine, is the intermediate product of L-tyrosine synthesis catecholamine in vivo.As medicine, general levodopa by name, has another name called Pardopa, and commodity breath by name is peaceful, Parcopa, Atamet, Stalevo, madopar, Prolopa etc.
Attached: left-handedly to refer to that the enantiomorph of organic compound rotates to inhour or clockwise direction making light in polarized light respectively with dextrorotation.Polarized light isomer that is left-handed or dextrorotation can be made to be called as levo form and dextrorotatory form.In organic chemistry, usually represent left-handed with (+), (-) represents dextrorotation.As the levotartaric acid of L-configuration, its full name can write L (+)-tartrate.
L-DOPA is antiparkinsonian drug, is applicable to primary parkinsonism and non-medicine originality Parkinsonism.L-DOPA enters cerebral tissue by hemato encephalic barrier, plays a role.Nausea and vomiting, poor appetite and the untoward reaction such as insomnia, illusion is had after L-DOPA takes.
2, physico-chemical property
English another name: 3-(3,4-Dihydroxyphenyl)-L-alanine, L-3-(3,4-Dihydroxyphenyl) alanine
Chemical name: L-Dopa
Molecular formula: C 9h 11nO 4
Molecular weight: 197.19
CAS accession number: 59-92-7
EINECS accession number: 200-445-2
Specific rotatory power-11.7 & ordm; (c=5.3,1N HCl)
Fusing point: 295 DEG C
3, pharmaceutical analysis
L-DOPA produces chemosynthesis, extracted form natural plant and microbial enzyme conversion method three main paties, natural levodopa derives from the seed of pulse family lamb's-quarters bean plant dragon's paw lamb's-quarters beans Stizotobium ochinchinensis (Lour) .Tang etWang, also can be synthesized by TYR in mammalian body He in brain.
(1) method name: the mensuration-nonaqueous titrations of levodopa bulk drug-levodopa.
Range of application: present method adopts the content of levodopa in titration measuring levodopa bulk drug.
Present method is applicable to levodopa bulk drug.
Method And Principle: trial-product adds Glacial acetic acid and shakes up, add Viola crystallina indicating liquid 2 after adding anhydrous formic acid dissolving, solution is titrated to aobvious green with perchloric acid titration liquid, and the result blank test of titration is corrected, according to titrating solution usage quantity, calculate the content of levodopa.
Reagent: 1. Glacial acetic acid, 2. anhydrous formic acid, 3. perchloric acid titration liquid (0.1mol/L), 4. Viola crystallina indicating liquid, 5. benchmark Potassium Hydrogen Phthalate.
Prepared by sample:
1. perchloric acid titration liquid (0.1mol/L)
Preparation: get anhydrous Glacial acetic acid (calculate by water content, every 1g water adds aceticanhydride 5.22mL) 750mL, add perchloric acid (70 ~ 72%) 8.5mL, shake up, let cool, add anhydrous Glacial acetic acid and make into 1000mL in right amount, shake up, place 24 hours.If survey the easy acetylize of trial-product, then must measure the water content of this liquid with aquametry, then be 0.01% ~ 0.2% by the water content that water and aceticanhydride are adjusted to this liquid.
Demarcate: be taken at 105 DEG C of benchmark Potassium Hydrogen Phthalates being dried to constant weight and be about 0.16g, accurately weighed, add anhydrous Glacial acetic acid 20mL and make dissolving, add Viola crystallina indicating liquid 1, be slowly titrated to blueness with this liquid, and titration results blank test is corrected.Every 1mL perchloric acid titration liquid (0.1mol/L) is equivalent to the Potassium Hydrogen Phthalate of 20.42mg.According to the consumption of this liquid and the amount of taking of Potassium Hydrogen Phthalate, calculate the concentration of this liquid.
2. Viola crystallina indicating liquid
Get Viola crystallina 0.5g, add Glacial acetic acid 100mL and make dissolving.
Operation steps: precision takes trial-product and is about 0.1g, add anhydrous formic acid 2mL and make dissolving, add Glacial acetic acid 20mL and shake up, add Viola crystallina indicating liquid 2, be titrated to solution with perchloric acid titration liquid (0.1mol/L) aobvious green, and the result blank test of titration is corrected.Every 1mL perchloric acid titration liquid (0.1mol/L) is equivalent to the C9H11NO4 of 19.72mg.
Note: " precision takes " mean take weight should accurately to take the thousandth of weight." precision measures " means that the accuracy measuring volume should meet the accuracy requirement to this volume transfer pipet in national standard.
Reference: Pharmacopoeia of People's Republic of China, Chinese Pharmacopoeia Commission compiles, Chemical Industry Press, version in 2005, one, p.85.
(2) method name: the mensuration-spectrophotometry of levodopa capsule-levodopa
Range of application: present method adopts the content of levodopa in spectrophotometry levodopa capsule.
Present method is applicable to levodopa capsule.
Method And Principle: trial-product adds hydrochloric acid soln and makes test liquid, puts ultraviolet-visible pectrophotometer, measures optical density in 280nm wavelength place, calculates its content.
Reagent: hydrochloric acid soln
Plant and instrument: ultraviolet-visible pectrophotometer
Prepared by sample:
1. hydrochloric acid soln gets hydrochloric acid 9mL, adds water and makes into 1000mL in right amount, shake up.
2. the content under content uniformity item is got in the preparation of need testing solution, and mix, precision takes in right amount (being about equivalent to levodopa 30mg), put in 100mL measuring bottle, add hydrochloric acid soln appropriate, jolting makes levodopa dissolve, and is diluted to scale with hydrochloric acid soln, shake up, filter, precision measures subsequent filtrate 10mL and puts in another 100mL measuring bottle, adds hydrochloric acid soln and is diluted to scale, shake up, obtain need testing solution.
Note: " precision takes " mean take weight should accurately to the thousandth taking weight." precision measures " means that the accuracy measuring volume should meet the accuracy requirement to this volume transfer pipet in national standard.
Operation steps: get need testing solution according to ultraviolet spectrophotometry, measure optical density in wavelength 280nm place, be 141 calculating by the uptake factor (E1%1cm) of C9H11NO4, obtain final product.
Note: spectrophotometry to prepare same batch of solvent of trial-product for contrast, should adopt the silica cuvette of 1cm.Using the maximum wavelength of optical density as mensuration wavelength, the optical density reading of general trial-product is less with the error between 0.3 ~ 0.7.The slit wavestrip width of instrument should be less than the half-width of trial-product absorption band, otherwise the optical density recorded is on the low side.The selection of slit width, should to reduce slit width time trial-product optical density no longer increase and be as the criterion.Because cuvette and solvent itself may have blank absorption, blank reading should be deducted after therefore measuring the optical density of trial-product, then calculate content.
Reference: Pharmacopoeia of People's Republic of China, Chinese Pharmacopoeia Commission compiles, Chemical Industry Press, version in 2005, one, p.86.
4, pharmacological toxicology
This product is for intending Dopaminergics antiparkinsonism drug, levodopa is the precursor substance of synthesis Dopamine HCL in body, and itself is parmacodynamics-less activity also, enters maincenter by hemato encephalic barrier, change into Dopamine HCL through dopa decarboxylase effect and play pharmacological action, improving Parkinsonian symptoms.Because this product can increase the neurotransmitter such as Dopamine HCL and norepinephrine in brain, can also improve the tolerance of brain to ammonia, and be used for the treatment of hepatic coma, improve maincenter function, patient is regained consciousness, and symptom is improved.
(1) anti-parkinson levodopa changes Dopamine HCL in brain, supplements the deficiency of Dopamine HCL in striatum, thus has the curative effect of anti-parkinson.Research shows, once used the patient of a large amount of levodopa treatment, after death in striatum, dopamine concentration is higher than non-medication curer 5 ~ 8 times; And dopamine concentration is consistent with the curative effect of levodopa in brain.Illustrate that in patient's nigro-striatal path, remaining dopaminergic neuron still has the ability storing Dopamine HCL.Its striatum dopa decarboxylase still has enough enzymic activitys that levodopa can be made to change Dopamine HCL into.
After levodopa treatment, the patient of about 75% obtains good therapeutic effect.Treatment initial response is more aobvious.The action character of levodopa is:
1. to mild and comparatively young patient curative effect is better, and severe and old debilitated patient's weak curative effect;
2. better to muscular rigidity and dyskinesia curative effect, and to muscular tremor symptom weak curative effect, as long-term prescription and larger dose still can take effect to the latter;
3. act on comparatively slow, often need medication within 2 ~ 3 weeks, just to occur the improvement of objective sign, within more than 1 ~ 6 month, just obtain greatest treatment efficacy, but persistent, and extend with administration time and increase progressively.
The parkinson's syndrome that levodopa causes other reasons is also effective.But invalid to caused by the antipsychotic drugs such as phenothiazines, because these medicines have the effect blocking central dopaminergic receptor.
(2) the pseudo-mediator theory that treatment hepatic coma hepatic coma pathogenesis is right is thought, all oxidized removing toxic substances in liver of normal body protein metabolism product phenylethylamine and tyrasamine.During hepatic insufficiency, in blood, phenylethylamine and tyrasamine raise, and generate pseudo-mediator respectively, Phenylethanolamine and octopamine (Octopus amine in neurocyte through B-hydroxylase), they instead of normal mediator---and norepinephrine, hinders neural function.Norepinephrine can be changed in brain with levodopa.Normal neuronal activity is recovered, and patient can be transferred to by stupor and reviving.Because can not liver function be improved, act on just temporary.
5, drug use
L-DOPA is antiparkinsonian drug.Enter cerebral tissue by hemato encephalic barrier, be transformed into Dopamine HCL through dopa decarboxylase decarboxylation, play a role.
L-DOPA is used for primary parkinsonism and non-medicine originality Parkinsonism, and centering, slight effect are better, severe or the elderly poor.
(1) usage and consumption
Preparation specification: tablet or capsule: 0.25g.
Oral: Parkinsonism, start 0.25 ~ 0.5g/ time, 3 ~ 4 times/day, increased by 0.125 ~ 0.5g every 3 ~ 4 days.Maintenance dose 3 ~ 6g/ day, point 3 ~ 4 clothes, feeling sick as occurred, should stop increment, increasing after transference cure again in dosage escalation process.As use same with dopa decarboxylase inhibitor, dosage can reduce by 50%.
(2) pharmacokinetics
L-DOPA is a kind of important biologically active substance in organism, Catecholamines Neurotransmitters in Blood Dopamine HCL, norepinephrine or adrenergic precursor, important intermediate from TYR to catechol or in melanic biochemical metabolism approach process, can directly (be called for short: COMT) change into 3-O-methyldopa and (be called for short: 3-OMD), generate Dopamine HCL through decarboxylation by catecholamine-O-methyltransgerase.This pathways metabolism is not present in healthy person body, but it is very important for being used for monitoring peripheral L-DOPA for the patient that disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase lack.
The pathways metabolism of L-DOPA is as follows:
Relevant English in above-mentioned pathways metabolism is explained as follows:
L-dopa: L-dopa;
S-adenosyl-L-metthionine:s-adenosine-L-Methionine;
S-adenosyl-L-L-homocysteine:s-adenosine-L-homocysteine;
L-dopa O-methyltransferase (Rn): Rn-Comt L-dopa oxygen position methyltransgerase;
3-O-methyl dopa:3-oxygen methyldopa;
2-oxoglutarate:2-ketoisocaproic;
L-glutamate: D-glutamicacid;
3-O-methyl dopa transaminase:3-oxygen methyldopa transaminase;
3-methoxy-4-hydroxy phenyl pyruvate:3-methoxyl group-4-hydroxyphenylphruvic acid;
Hypothetical: suppose;
NAD: NAD;
NADH: nicotinamide adenine dinucleotide reduced;
3-methoxy-4-hydroxy phenyl lactate:3-methoxyl group-4-hydroxyphenyl lactic acid;
Homovanillate: homovanillic acid.
By intestinal absorption after L-DOPA is oral.After oral levodopa, absorbed rapidly from small intestine by the active transport system of die aromatischen Aminosaeuren, about 0.5 ~ 2 hour, Plasma Concentration peaking, be distributed widely in each tissue in body, 1% enters maincenter changes into Dopamine HCL and plays a role, and all the other major parts all become Dopamine HCL in the outer metabolism decarboxylation of brain, therefore onset is slow.
Be degraded into Dopamine metabolites in 80% in 24 hours after L-DOPA is oral, be mainly homovanillic acid and dihydroxyphenyl acetic acid, by renal excretion; Small portion levodopa changes melanochrome (melannin) into; Some levodopa another (is called for short: COMT) methylate, change 3-methoxyl group DOPA into through catechol-O-methyl transferase (COMT); Above metabolite is drained rapidly by kidney.This metabolic process consumes more COMT, and in COMT reaction, methyl is mainly from the methionine(Met) in food, therefore long-term taking levodopa can cause methionine deficiency.
Some metabolite can make urine redden look.Prototype excretes about 5%, by lactation.
Its uptake rate is by various factors, there are other amino acid to compete with it active transport system (as high protein diet) etc. in as high in gastric emptying (with take cholinocepter blocking agent), gastric acidity or small intestine, all can reduce its bioavailability.After absorption, during first passage liver, major part is by decarboxylation, is transformed into Dopamine HCL.Also considerable part is had to be generated Dopamine HCL by decarboxylation in intestines, the heart, kidney.And Dopamine HCL is not easily through hemato encephalic barrier, therefore enter the levodopa of central nervous system less than 1% of consumption.In peripheral tissues, form a large amount of Dopamine HCL is the reason causing untoward reaction.
Plasma half-life (t 1/2) be 1 ~ 3 hour, as taken periphery dopa decarboxylase inhibitor (carbidopa) simultaneously, the consumption of levodopa can be reduced, make it the amount entered in brain and increase, and the untoward reaction that periphery Dopamine HCL causes can be reduced.
(3) untoward reaction
Gastrointestinal reaction, as Nausea and vomiting, poor appetite, sees the treatment initial stage, and uneasiness, insomnia, illusion mental symptom can appear in medication after 3 months, still can have postural hypotension irregular pulse and involuntary movement etc. in addition.
Untoward reaction is more.Be divided into early stage and long-term two large classes.
1. digestive tube: reaction incidence in this product independent medication upper gastrointestinal road is higher, apocleisis, Nausea and vomiting, poor appetite, abdominal discomfort, flatulence, dry, dysphagia, polysialia, tongue burning sensation, bitter taste, diarrhoea or constipation etc., make peptide ulceration worsen, cause bleeding.Along with the prolongation of administration time, GI adverse reaction rate reduces, but still has groups of people can not tolerate effective dosage.As share with decarboxylase inhibitor, then GI tolerance is better, especially with food with take, gastral disorder slightly and be transient, only has a little patient to add and uses antiemetic.
2. cardiovascular systems: orthostatic hypotension, palpitaition, tachycardia and hypertension.Also there is arrhythmia in some patient, mainly because the Dopamine HCL of new life acts on the cause of heart beta receptor.
3. central nervous system: grit one's teeth in tarantism sample and involuntary action, hand tremor increase, bradykinesia outbreak, sleep, ataxia, jerk, numbness, weakness, tired, headache, opisthotonus, entanglement, excitement, anxiety, glad, insomnia, nightmare, convulsions etc., phrenoplegia also has to be vainly hoped or illusion, major depression (comprising introgression) and hypomania.
4. blood system: hemolytic anemia, granulocytosis, oxyphorase and hematocrit value decline, oligoleukocythemia etc.
5. eye: blepharospasm, diplopia, blurring of vision, platycoria etc.
6. other: to bleed after rhinorrhea, flush, fash, urine and sweat blackout, many, the uroschesis of perspiring or incontinence, menstruation, oedema, the storehouse coomb's test Coomb positive, the rising of of short duration alkaline phosphatase, AST, pyruvate aminotransferase, serum lactic dehydrogenase, bilirubin and blood urea nitrogen can be had, urine protein, uric acid increase etc., occasionally can increase the weight of gout.Also can sexual function improving.
6, interact
1. benzene phenodiazine Bi class medicine and phenytoin Sodium can the Antiparkinsonian effects of antagonism this product, should note with the used time.
2. methyldopa or clonidine can the antidetonation of antagonism this product to quiver numbness effect, increase the weight of the side effect of its postural hypotension.This product should not with the two same use, also can not with phenothiazines and the general amine of methoxy-DDT fiber crops with using.
3. oxidase inhibitor can stop the degraded of Dopamine HCL in vivo, strengthens its effect, but can cause increased heart rate and hypertensive crisis, should not with this product with using.In oxidase inhibitor of stopping using with this product first two weeks, or inactive this product is after 2 days, uses oxidase inhibitor.
4. should not use together with serpentine and adrenomimetic drug class medicine.Serpentine can reduce the effect of this product, and adrenomimetic drug class medicine can increase the weight of cardiovascular untoward reaction.
5. GENERAL ANESTHETICS and this product are share, and easily cardiovascular accident occur, should before general anesthesia at least 1 day inactive this product.
6. vitamins B 6for dopamine decarboxylase enzyme prothetic group, can increase peripheral tissues's decarboxylase, heavy dose of application can reduce this product effect.Unsuitable and the vitamins B of this product 6with using.But as share with decarboxylase inhibitor, then vitamins B 6dopamine decarboxylase effect can be promoted in brain, strengthen the effect of levodopa.
7. this product can strengthen the antihypertensive effect of guanethidine and methyldopa, and they also can affect the Antiparkinsonian effect of this product simultaneously.
8. the extrapyramidal symptoms that causes of antipsychotic drug, unavailable this product treatment, because of both have antagonistic action.
9. with carbidopa, Benserazide, Symmetrel, kemadrin with there being synergy, consumption can be reduced and alleviate untoward reaction.
MK-485 (α-methyldopahydrazine) has two kinds of isomer, its levo form claims carbidopa (carbidopa) to be stronger L-aromatic amino acid decarboxylase inhibitor, owing to not easily passing through hemato encephalic barrier, therefore when share with levodopa, only can suppress the activity of periphery dopa decarboxylase, thus reduce the generation of Dopamine HCL in peripheral tissues, improve the concentration of Dopamine HCL in brain simultaneously.Like this, the curative effect of levodopa can be improved, the side effect of its periphery can be alleviated again, so be the important adjuvant of levodopa.Carbidopa is applied separately substantially without pharmacological action.By carbidopa and levodopa by 1: 10 dosage share, the effective dose of levodopa can be made to reduce 75%, benserazide (benserazide) has same effect with carbidopa, it and levodopa claim madopar (madopar) by the compound preparation that make at 1: 4, are applied to clinical.
Amantadine (amantadine) is former is antiviral drug, rear discovery its also have the effect of anti-parkinson, curative effect not as good as levodopa, but is better than cholinocepter blocking agent.Instant effect and holding effect is short, medication a couple of days can obtain greatest treatment efficacy, but after being used in conjunction 6 ~ 8 weeks, curative effect weakens gradually.Synergy has been share with levodopa.The mechanism of its anti-parkinson may be to impel complete dopaminergic neuron release Dopamine HCL remaining in striatum; And the re-uptake of Dopamine HCL can be suppressed; And have the effect of direct exciting Dopamine Receptors and more weak cholinolytic effect.After long-term prescription, there is livedo reticularis in common skin of lower extremity, may be to be discharged by catecholamine to cause caused by peripheral blood vessel contraction.Occasionally cause convulsions, therefore epileptic's forbidding.Every per daily dose, can induced insomnia, spirit uneasiness and ataxia etc. more than 300mg.
10. Propranololum can strengthen this product curative effect, also strengthens this product induction stimulating growth Secretion of Associative.
7, precaution
Easily there is " switch " phenomenon (patient suddenly many dynamic uneasinesses is for "ON" then occurs again that myotony motion can not be "Off") in age lighter patient, dosage or the upset of quiet note levodopa can be reduced or control this phenomenon, share curative effect with vitamin B6 or chlorpromazine etc. to reduce, increase curative effect is share with periphery dopa decarboxylase inhibitor carbidopa etc., reduce side effect, now can merge application vitamin B6 taboo and share with oxidase inhibitor, ephedrine, serpentine and adrenomimetic drug.Peptide ulceration, hypertension, psychosis, diabetes, irregular pulse and blood of angle closure glaucoma patients are forbidden.
1. forbid in weak to this product allergy, critical cardiovascular conditions, encephalosis, endocrine disturbance, psychosis, serious neurological, used the agent of monoamine oxydasis, narrow-angle glaucoma, peptide ulceration in 2 weeks, had convulsions history patient, and First Trimester pregnant woman, wet nurse, puerpera and less than 12 years old children.
2. hemolytic anemia, liver or ephrosis, have scheming infarct history, Peptic Ulcers, epilepsy, mental anomaly, tuberculosis, bronchial asthma and patient with gout to be cautious use of.
3. this product is as taken time on the feed, particularly high protein diet, can interference medicament by blood plasma to the transfer of nervus centralis.Medication and enter the fluctuation that low albumen fast food can reduce clinical response between two meals.
4. the degree of safety due to this product is very little, strictly should grasp indication, and detailed medical history-taking also checks.Dosage is determined according to the tolerance of patient, from low dose, increases gradually, until toxic reaction appearance and decrement maintain.The toxicity occurred should process in time.
5. early treatment often has asymptomatic orthostatic hypotension; Also someone occurs dizzy or faints, and continuing medication can take a turn for the better, and can tolerate after the general several months.During medication, should be slow when activity or change position.
6. intraocular pressure, routine blood test and with or without anaphylaxis should be noted; Should often to have a blood test sugar.
7. this product is to the generally display 2 ~ 3 weeks time of the effect of Parkinsonism.Shake the aggravation of quite property paralysis period, continue 1 ~ 3 hour outbreak in 1 minute, and show effect at one time every day, represent that consumption is too large or be subject to caused by emotional shock.
8. when the abnormal movement of involuntary muscle appears in patient, should decrement immediately, otherwise symptom will be aggravated.
9., during medication, should avoid eating as far as possible and be rich in B 6food, as yeast, whole wheat, wheat bran, internal organ and lean meat, vegetables with green leaves; Be not engaged in the activity that band is dangerous, as driven, ascending a height; When being in a bad mood, the change of intelligence, the aspect such as behavior time, answer decrement.
10., during medication, false uric acid in the false glucose in urine positive, the urine ketoboidies positive and urine can be had to raise.
8, clinical study
[effect] carbidopa and levodopa share treatment paralysis agitans.
The composite sheet of [chemical composition] new drug " carbidopa sheet (small pieces) " and old medicine " levodopa sheet (sheet) " combination packaging, the every sheet of small pieces is containing carbidopa 25mg, and large stretch of every sheet is containing levodopa 0.25g.
[pharmacological action] Kabaduoba can suppress the levodopa decarboxylation of periphery to become Dopamine HCL, it can not through hemato encephalic barrier, thus make to enter the levodopa of low dosage in brain by decarboxylation, reach the Dopamine HCL of effective concentration, and reduce the side effect of periphery Dopamine HCL.
[drug interaction] and oxidase inhibitor are share and can be caused hypertension.
With vitamins B 6, chlorpromazine or Papaverine share and can reduce this product effect.
Same with strengthening this product effect with o-methyl-diphenhydramine.
Vitamins B 6week is metabolized to Dopamine HCL outside can to accelerate levodopa.
This product and tricyclic antidepressant share and may produce hypertension and dyskinesia, should focus on.
After thiodiphenylamine, butylbenzene mountain amine, phenytoin Sodium and Papaverine and levodopa merge and apply, there is the report that levodopa curative effect reduces.
[untoward reaction] carbidopa is without special untoward reaction, though its adverse reaction rate that can reduce alone levodopa and produce, but when taking carbidopa and levodopa, the untoward reactions such as dry, constipation, Nausea and vomiting, dizziness, many dynamic, palpitaition, salivation still can be there is.And the maincenter untoward reaction that this product can not alleviate levodopa occurs, as involuntary movement, dyskinesia, mental anomaly etc.
[contraindication] following patient's forbidding carbidopa and levodopa sheet: the patient that the metabolisms such as cardiovascular, internal secretion, blood, liver, lung and kidney are incomplete, the patient of sympathomimetic amine, narrow angle glaucoma patient.Lactating women, pregnant woman are cautious use of.
[product specification] be levodopa 200mg 1., carbidopa 50mg; 2. levodopa 100mg, carbidopa 25mg.
[usage and dosage] is oral.When usual amounts starts one day with carbidopa 25 ~ 50mg, levodopa 0.25 ~ 0.5g, (one day with 1 ~ 2 little lattice dress); After treating one week, often take 3 ~ 4, increase carbidopa 25mg, levodopa 0.25g, (with 1 little lattice dress); Or follow the doctor's advice.
Maintenance dose carbidopa 75 ~ 100mg on the one, levodopa 0.75 ~ 1g (3 ~ 4 little lattice dress) point take for 3 ~ 4 times.Singly taking the patient of levodopa sheet, as carbidopa and levodopa sheet will be taken, should stop taking levodopa sheet and just can take this product at least 8 hours later, when taking, the predose of its levodopa should be equivalent to 25% of former alone dosage, then dosage maximal dose every day carbidopa must not more than 0.2g gradually again, and levodopa must not more than 2g (namely must not fill more than 8 little lattice).
[storage practice] shading, airtight preservation.
[precaution] alone carbidopa is invalid, must share with levodopa.Use oxidase inhibitor person simultaneously, start will stop using with fortnight before this product, cause during cone vitro reactions should not use at medicine.
(2) spermidine/spermine N 1the research overview of-Transacetylase
Cohen and Morgan was at report in 1998: SSAT is extensively present in mammalian tissues, eliminated spermine at endocellular metabolism, but was in pole low-level at SSAT normally or in the mammalian tissues of not inducing.SSAT is the rate-limiting enzyme for Polyamine catabolism, and the reverse of catalysis spermine turns to spermidine and spermidine reverse turns to putrescine, can promote that polyamines is degraded, drains, circulate and/or cell internal recycle.
Casero & Pegg can be induced by multi-medicament, somatomedin, polyamines, polyamine analogs, toxicant, hormone and physiological stimulation at report SSAT in 1993 and produce, but induces the period produced different for each individuality.The expression regulation of SSAT can occur in transcribe, mRNA stability, mRNA translation and the level of protein stabilization.
SSAT gene comprises polyamines response composition at transcription initiation site 1522-1492, and in these 31 pairs of basic sequences, polyamines reaction is confirmed as 9 pairs of basic sequences.By polyamine analogs N 1, N 12the SSAT of the polyamines reacted constituent mediation of-bis-(ethyl) spermine or natural polyamines induction transcribes.
In rat hepatocytes, SSAT molecular weight is less than 1000; be equivalent to be induced cell 60; 000 molecule, induces mammalian tissues that SSAT level can be caused to improve by different inductor, thus can as the potential acetylize individuality of main (former) amine-containing compound.
Normally there is sufficient SSAT enzyme in the induction of SSAT or overexpression, or use the centrifugal supernatant liquor of 100,000Xg can reach (Casero & Pegg1993 in testing in vitro in cell; Fogel-Petrovie1997; Matsui & Pegg1980; Pietila1997).
SSAT a kind of promotes substrate acetyl enzyme, particularly acetyl spermine and spermidine or its analogue.Current SSAT active level method depends on this area hi-tech personnel and complicated experimental technique.More particularly, needing a kind of quantivative approach by detecting SSAT activity to spermidine/spermine substrate acetyl form, comprising by detecting amantadine and L-3,4 dihydroxyphenylalanine, thus can be used as to detect different pathological conditions.
In a word, by literature search etc. existing technical information analysis shows, up to the present, not yet finds that there is the report of the aspect such as acetylize measuring method of the activity of SSAT, enzyme kinetics and SSAT spermidine and spermine substrate.
Summary of the invention
The technical problem that will solve required for the present invention discloses a kind of detection Mammals spermidine/spermine N 1the method of-acetyltransferase activity, namely assesses in potential SSAT path and mainly or only regulates the acetylizad specific substrate of N-by SSAT, to overcome the above-mentioned defect that prior art exists.
That is, the present invention is by the research such as experimentation on animals, clinical trial and theory study, and one of object is intended to provide-kind of new quantitative technique spermidine/spermine substrate acetyl form being detected to SSAT activity.
Two of object of the present invention assesses in potential SSAT path mainly or only to regulate the acetylizad specific drugs material standed for of N-by SSAT.
(1) technical conceive
Contriver through the latest find of research is: L-DOPA is a kind of important biologically active substance in organism, Catecholamines Neurotransmitters in Blood Dopamine HCL, norepinephrine or adrenergic precursor, important intermediate from TYR to catechol or in melanic biochemical metabolism approach process, can directly (be called for short: COMT) change into 3-O-methyldopa and (be called for short: 3-OMD), generate Dopamine HCL through decarboxylation by catecholamine-O-methyltransgerase.This pathways metabolism is not present in healthy person body, but it is very important for being used for monitoring peripheral L-DOPA for the patient that disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase lack.
The part metabolism of levodopa can produce acetylize metabolite N-acetyl base-L-3,4 dihydroxyphenylalanine by the induction of SSAT; SSAT activity can be analyzed by the acetylize metabolite N-acetyl base-L-3,4 dihydroxyphenylalanine detecting SSAT substrate levodopa; and being proportionate of the rise of SSAT activity and cancer; therefore; the cancer markers that L-3,4 dihydroxyphenylalanine can regulate as SSAT, in this, as an index of early prevention, diagnosis, detection, protection, treatment and research cancer in medical treatment and scientific research.
Further research detects the quantitative technique of SSAT activity to spermidine/spermine substrate acetyl form; improve medical diagnosis quality and the scientific research effect of cancer; realize early discovery, early diagnosis, early treatment, avoid and alleviate the burden of family and society, be conducive to the life quality of raising patient.
The patient that disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase lack particularly disturbances in patients with Parkinson disease gets more and more; have a strong impact on health and the life quality of Chinese population; white elephant is brought to family and society; development prevention, diagnosis, detect, product that protection, treatment and research are conducive to the aspects such as the patient that disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase lack; particularly medicine, diagnostic reagent and healthcare products etc., have significant social benefit, economic benefit.
According to this idea and thinking, contriver, by experimental study repeatedly and analysis, successfully obtains result of study and the application product of expection.
(2) for detecting spermidine/spermine N 1the method of-acetyltransferase activity
The invention provides a kind of detection Mammals spermidine/spermine N 1the method of-acetyltransferase activity, assesses in potential SSAT path and mainly or only regulates the acetylizad specific substrate of N-by SSAT.
The present invention regulates the acetylizad specific substrate of N-to carry out many-sided test and study in prevention, diagnosis, detection, protection, treatment and research etc. to SSAT.According to the data description of invention and reference in vivo, measure the analytical procedure of SSAT activity in external model.
The present invention provides following technical scheme according to above-mentioned technical conceive and result of study.
First the present invention will assess in potential SSAT path and mainly or only regulate the acetylizad specific drugs material standed for of N-by SSAT, and concrete grammar is as follows:
1) substrate, inhibition solution and tissue or 37 DEG C, the cell solubleness evaluation of hatching altogether;
2) substrate, the tissue of inhibition solution to cryopreservation or the toxicity assessment of cell;
3) comprise application NAT1, NAT2 path specific inhibitor according to the method establishment external model of CYP path phenotype in the external drug metabolism guide of U.S. FDA 2007, confirm the specificity of NAT1, NAT2 selective depressant, in table 1.
Table 1, SSAT regulate the acetylizad specific drugs material standed for of N-
For detecting the N of Mammals spermine/spermine 1the method of-acetyltransferase activity, it comprises the following steps:
1) Mammals a certain amount of a kind of medicine of hatching (that is: SSAT substrate);
2) analytic samples such as certain tissue, cell or body fluid are obtained from Mammals;
3) detecting the acetylize metabolite in analytic sample, there is being associated (positive correlation) with SSAT activity in acetylize metabolite.
The present invention is achieved through the following technical solutions:
1) Mammals described in comprises people or other animals;
Other described animals, comprise in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or rhesus monkey etc. one or more;
One or more in preferred people, mouse, rat, monkey, pig, rabbit or dog etc., one or more further preferably in people, rat or monkey etc.;
Described hatching, is referred to and to be hatched altogether by medicine (that is: SSAT substrate) and mammalian tissues, cell or body fluid;
Described medicine (that is: SSAT substrate) is L-3,4 dihydroxyphenylalanine, the dosage range of SSAT substrate: 1 ~ 10mg/kg, preferably 3 ~ 6mg/kg;
2) analytic sample described in be derive from mammiferous humoral sample or cell sample that 0 ~ 24h hatches altogether one or more, preferably 10 ~ 60min mammalian cell of hatching altogether, mammalian cell preferred rat hepatocytes further;
Described humoral sample be comprise in blood, urine or saliva one or more;
The preferred L-3,4 dihydroxyphenylalanine of mammiferous humoral sample that 0 described ~ 24h is hatched altogether enters in mammalian body and collects urine specimen in 2 ~ 24h, preferably L-3,4 dihydroxyphenylalanine enters in mammalian body and collects urine specimen in 8h further, preferably L-3,4 dihydroxyphenylalanine enters in mammalian body and collects urine specimen in 4h again, is most preferably that L-3,4 dihydroxyphenylalanine enters 2h interior collection urine specimen in mammalian body;
3) in analytic sample, the existence of acetylize metabolite is an indication of Mammals SSAT activity, and the existence of acetylize metabolite is associated (positive correlation) with SSAT activity;
Acetylize metabolite method in described detection analytic sample is the measured quantity according to acetylize metabolite in Mammalian samples, make typical curve to determine the activity of SSAT in mammalian body, can by multiple technologies be comprise in chromatographic detection method, radio-labeling, mass spectrum, infrared, near infrared or ultraviolet detection etc. one or more;
Described chromatographic detection method be comprise gas-chromatography, high performance liquid chromatography (is called for short: one or more HPLC), in thin-layer chromatography, specific antibody chromatogram or antibody affinity chromatography etc.;
Described positive correlation refers to that the measured quantity of acetylize metabolite is higher, and the activity of SSAT in mammalian body is higher.
Specific experiment process of the present invention is as follows:
1, materials and methods:
DMSO), water, formic acid, hydrochloric acid, trypan blue solution, tetramethyl-azo azoles salt, Krebs-Henslaite damping fluid reagent: methyl alcohol, dimethyl sulfoxide (DMSO) (are called for short:.
1.1 substrate: amantadine, spermidine, para-aminosalicylic acid, PABA, sulfamethoxazole (are called for short: SMZ).
1.2NAT1 and NAT2 pathway inhibitor: (±) methotrexate, paracetamol, Quercetin.
1.3N-acetylize metabolite: N-ethanoyl-amantadine (AA), N-ethanoyl-sulfamerazine.
Mark in 1.4: N-ethanoyl-d 3-amantadine, N-ethanoyl-sulfamerazine-d 4.
2, the former liver cell preparation of freezing female mouse
Adopt rat liver cells, cell adopts Celsis ex vivo technique to obtain, stored under refrigeration in liquid nitrogen.Use cryopreserved hepatocytes water-bath thawed the day before yesterday, make suspension with substratum, centrifugal, (be called for short: KHB) suspendible again, as liver cell storing solution with Krebs-Hensleite damping fluid;
Before induction experiment, hepatocellular concentration and active employing determination of trypan blue staining rat hepatocytes activity.Cell storing solution adds KHB and adjusts cell concn to target level;
First rat hepatocytes was measured with trypan blue exclusion active before plating.
3, solution preparation
Prepared by substrate solution: para-aminosalicylic acid, para-amino benzoic acid, sulfamerazine, amantadine, spermidine, L-3,4 dihydroxyphenylalanine;
Prepared by inhibitor solution: (±) methotrexate, paracetamol, Quercetin.
(1) solubleness evaluation
In substrate, the inhibitor aqueous solution, add KHB, add or do not add DMSO, measure its final concentration.
Prepare each chemical storing solution, be then diluted to test fluid with preheating KHB.Test fluid is prepared into 3 concentration, and HPLC/UV measures its optical density, makes typical curve, obtain linear regression relation with optical density-concentration
(2) mtt assay rat hepatocytes cytotoxicity detects
Mtt assay is adopted whether to have cytotoxicity to the former hepatocytes of refrigeration for detection substrate and inhibitor when detectable level.
Adopt 96 orifice plates, liver cell respectively with substrate, the material standed for of different concns, add NAT1 or NAT2 or NAT1 and NAT2, altogether hatching simultaneously.Then preincubate, every hole adds the KHB containing equivalent MTT, then hatches.After hatching, remove substratum, add DMSO and dissolve, then use deck vibrator hydrotropy, measure the optical density of solution with microplate reader 540nm.
(3) rat hepatocytes N-acetylize is measured
Adopt 24 well culture plate vitro culture, each substrate or material standed for and NAT1 or NAT2 inhibitor are hatched separately or hatch altogether, parallel in duplicate.
Add certain density inhibitor or solvent control to culture plate.Then each hole adds active Hepatocyte storing solution, hatches altogether.Then preincubation, adds the mixture of each substrate, material standed for or surrogate respectively, continues to hatch.At the end of cultivation, in every hole, add ice methyl alcohol stopped reaction.
Due to solubleness restriction, L-DOPA is independently cultivated, instead of mixes with other four material standed fors.Parallel two aliquot sample in every hole, before LC/MS and LC/MS/MS analyzes potential N-acetylize metabolite, are stored in bottle and preserve.
(4) LC/MS and LC/MS/MS method is to the bioanalysis of N-acetylize metabolite
This research adopts the N-acetylize metabolite of amantadine and sulphamethazine in LC/MS/MS quantitative analysis rat hepatocytes sample.N-acetylsulfanilamide diformazan pyrimidine and N-acetyl amantadine are respectively with N-acetylsulfanilamide diformazan pyrimidine-D 4with N-acetyl-D 3-amantadine is interior mark.Calibration standard: N-acetyl amantadine, N-acetylsulfanilamide diformazan pyrimidine.QC sample concentration is respectively: N-acetyl amantadine 0.2,80 and 160ng/ml, N-acetylsulfanilamide diformazan pyrimidine 2800,1600ng/ml.
Due to this research carry out time, there is no available metabolite reference standard, thus with target spot metabolite know method for distinguishing monitored spermidine, para-aminosalicylic acid, para-amino benzoic acid, L-3,4 dihydroxyphenylalanine N-acetylize metabolite.Deriving from In Cultured Rat liver cell sample first for scanning medicine prototype, then carrying out the scanning of potential N-acetylize metabolite, the precursor of parent scanning characteristic fragment, carries out directed multiple-reaction monitoring simultaneously.
4, results and analysis
(1) drug candidate, substrate and inhibitor are water-soluble
Have evaluated water-soluble when liver cell incubation buffer reaches target final concentration of the substrate in KHB damping fluid, inhibitor.Dimethyl sulfoxide (DMSO), methyl alcohol or 0.5MHCl hydrotropy can be added when medicine is soluble, assess solubility promoter by linear UV absorption process detection experiment concentration.Amantadine, spermidine are solvable.
(2) cytotoxicity of cryopreserved primary rat hepatocyte
Substrate adds or do not add NAT1, NAT1/NAT2 inhibitor does not have cytotoxicity to hepatocytes, except amantadine is at maximum concentration 2645 μMs (having cytotoxicity).Three inhibitor, only have (±) methotrexate, (±) methotrexate and acetparaminosalol phenol mixture, or Quercetin does not have cytotoxicity in test concentrations to rat hepatocytes yet.
(3) the acetylizad positive control of the N-of NAT1 and NAT2 in rat hepatocytes
NAT1 specific inhibitor Rheumatrex concentration 50 μMs, NAT2 specific inhibitor acetaminophen concentration 2144 μMs, at the end of hatching, N-acetylize is about 0.15%.
Inhibition NAT1 and NAT2 that be formed in of metabolite coexists 60min suppressed 28%.When there being NAT1 and NAT2 inhibitor to exist, 54%N-acetylize is suppressed.
(4) LC/MS/MS quantitative analysis N-acetyl-sulphamethazine and N-ethanoyl-amantadine
N-acetyl-sulphamethazine and ethanoyl-amantadine quantitative analysis adopt N-acetylsulfanilamide diformazan pyrimidine-D 4with N-acetyl-D 3-amantadine is interior mark.N-ethanoyl-amantadine correcting range 0.1 ~ 200ng/ml, N-acetyl-sulphamethazine correcting range 1 ~ 2000ng/ml.The matched curve of LC/MS/MS quantitative analysis correction is origin-excluded quadratic regression equation, and all concentration uses 1/x weighting factor.
N-acetyl-sulphamethazine:
The coefficient of determination: R^2=0.998133
Calibration curve :-1.4497e-007*x^2+0.00454808*x+6.02654e-005
N-ethanoyl-amantadine:
The coefficient of determination: R^2=0.999811
Calibration curve: 2.81933e-006*x^2+0.112015*x+0.00309319
The calibration standard curve of N-acetyl-sulphamethazine and N-ethanoyl-amantadine is Fig. 1, Fig. 2 respectively.
(5) the N-acetylize of rat hepatocytes SSAT positive control
SSAT substrate two positive controls are adopted: amantadine and spermidine in research.In rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor share, or the common specific inhibitor of NAT1 and NAT2, independently assess N-acetylizing.
NAT1 specific inhibitor methotrexate concentration is 50 μMs.NAT2 specific inhibitor acetaminophen limits due to solubleness, and concentration is 2144 μMs.The common inhibitor quercetin concentration of NAT1 and NAT2 is 480 μMs.In rat hepatocytes sample, N-acetylize metabolite derives from rat hepatocytes and amantadine (267 μMs and 2670 μMs) is hatched altogether, and N-acetyl metabolite amount is too low and could not detect.Concentrated rat hepatocytes and 267 μMs of amantadines are hatched sample and N-ethanoyl-amantadine still can not be detected.And amantadine residuum has almost no change when hatching.Result is consistent with BRI No.BIM-2009-001 result of study.In rat urine, low-level N-acetylize-amantadine is checked through in vivo in research.
Former medicine residuum ratio is calculated by following formula:
Detected result shows: the N-acetylize metabolite of spermidine, forms appearance fast at 0.At the end of hatching, quantity is close to 14% of the former medicine quantity added.This acetylization reaction does not suppress because of NAT1 inhibitor (50 μMs of methotrexates) and NAT2 inhibitor (2144 μMs of acetaminophen).But 26% acetylization reaction is suppressed when there is merely NAT1 inhibitor (50 μMs of methotrexates), use 480 μMs of Quercetins also can produce restraining effect, inhibitor acts on NAT1 and NAT2 two paths.Former medicine is residual not to be changed.Meta-bolites, metabolism suppress, the remaining discordance of former medicine can be solved by quantitative analysis.
SSAT substrate positive control and NAT1 and NAT2 inhibitor test-results show SSAT substrate spermidine N-acetylize can occur in female rats hepatocyte model.The effect of N-ethanoyl is not combined suppression (methotrexate 50 μMs and acetaminophen 2144 μMs) by NAT1 inhibitor and NAT2 inhibitor.But application methotrexate 50 μMs or acetaminophen 2144 μMs but have impact separately.Quercetin adopts concentration 480 μMs, in so high concentration, non-specific retarding effect may occur.In this research, confirm that Optimized model parameter comprises inhibitor concentration further, be one of this project undeveloped reserve.
(6) acetylizing of rat hepatocytes substrate L-DOPA
SSAT substrate material standed for L-DOPA is adopted in research; in rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor share; or the common specific inhibitor of NAT1 and NAT2, independently assess N-acetylizing.
Result shows: acetylize metabolite increases along with incubation time extends generation.When the L-3,4 dihydroxyphenylalanine added is 200 μMs, produce 0.5% metabolite; When the L-3,4 dihydroxyphenylalanine added is 20 μMs, producing the metabolite of 5.2%, there is the saturated or self-retarding effect of reaction in prompting.When the L-3,4 dihydroxyphenylalanine added is 20 μMs, former medicine residual quantity is lower.There is the common inhibitor (Quercetin of NAT1 and NAT2,480 μMs) when existing, metabolite is formed significantly suppressed (> 90%), and NAT1 inhibitor (methotrexate, 50 μMs) and NAT2 inhibitor (acetaminophen, 2144 μMs) exist, metabolite is formed not suppressed.
(7) conclusion
Show from above result, can produce NAT1 and NAT2 substrate N-acetylizing in female rats hepatocyte model, application NAT1 inhibitor methotrexate and the acting in conjunction of NAT2 inhibitor acetaminophen can suppress N-acetylization reaction.On the contrary, there is the N-ethanoyl effect that can not change SSAT substrate spermidine in these two inhibitor, also demonstrate the specificity of these two NAT inhibitor simultaneously.
Result also shows L-3,4 dihydroxyphenylalanine and is better than NAT1 and NAT2 to SSAT selectivity, and the known undetected acetylizing of SSAT substrate amantadine may be because sample is different.SSAT raises and being proportionate of cancer, therefore the L-3,4 dihydroxyphenylalanine cancer markers that can regulate as SSAT.
(4) technology speciality
The present invention prevents, diagnoses, detects, protects, treats and studies tumour to provide a kind of new detection method; namely mainly or only regulating the acetylizad specific substrate of N-by SSAT by assessing in potential SSAT path, having found a kind of detection Mammals spermidine/spermine N 1the method of-acetyltransferase activity, thus find in the morning of tumour, early carried out improvement in diagnosis and early treatment etc., improve, to overcome the above-mentioned defect that prior art exists.
That is; the present invention is by the research such as experimentation on animals, clinical trial and theory study; provide a kind of quantitative technique spermidine/spermine substrate acetyl form being detected to SSAT activity newly; the method of SSAT activity is analyzed by the acetylize metabolite detecting SSAT substrate; wherein: SSAT substrate is levodopa, the part metabolism of levodopa can produce acetylize metabolite N-acetyl base-L-3,4 dihydroxyphenylalanine by the induction of SSAT.L-3,4 dihydroxyphenylalanine is better than SSAT selectivity and raises and being proportionate of cancer NAT1 and NAT2, SSAT, therefore the L-3,4 dihydroxyphenylalanine cancer markers that can regulate as SSAT.
The inventive method is safe and effective, and practicality is comparatively strong, and its working method is convenient and swift, easy to use, and positive rate is high, Be very effective, can be used for preventing, diagnosis, detect, protection, treatment and study of various tumour supplementary means.
The present invention studies cancer markers targetedly; find a kind of new detection method; make beyond thought achievement; its use safety; prevention, diagnosis, detect, protection, treatment and the Detection results of study of various tumour obvious; and use range is wide especially, therefore easily applies, can have a tremendous social and economic benefits in the short period of time.
In a word; active adaption of the present invention modern medical service and the need of work of scientific research field and the needs of human nature service; the present invention is that research and development antitumorly provide new detection means; having important value to improving existing medical level, is for preventing, diagnosing, detect, protect, treat and the detection method of the aspect such as Effect of Anti tumour and directly related disease thereof.
Accompanying drawing explanation
Fig. 1 is N-ethanoyl in cryopreserved primary rat hepatocyte-sulphamethazine LC/MS/MS quantitative correction typical curve;
Wherein, × representing standard substance, ◇ represents gas phase sample;
Compound name: compound name; AA: ethanoyl SMZ;
Coefficient of determination: the coefficient of determination; Calibration curve: calibration (demarcation) curve;
Ref: reference; Area: area;
Conc: concentration; Response type: response type;
Internalstd: internal standard is poor; Curve type: curve type;
Origin: initial point; Exclude: get rid of;
Weighting: weighting; Axis trans: axis shifts;
None: nothing; Response: reaction;
Ng/ml: nanograms/milliliter.
Fig. 2 is N-ethanoyl in cryopreserved primary rat hepatocyte-amantadine LC/MS/MS quantitative correction typical curve;
Wherein, × representing standard substance, ◇ represents gas phase sample;
Compound name: compound name; AA: acetylized admantadine;
Coefficient ofdetermination: the coefficient of determination; Calibration curve: calibration (demarcation) curve;
Ref: reference; Area: area;
Conc: concentration; Response type: response type;
Internalstd: internal standard is poor; Curve type: curve type;
Origin: initial point; Exclude: get rid of;
Weighting: weighting; Axis trans: axis shifts;
None: nothing; Response: reaction;
Ng/ml: nanograms/milliliter.
Fig. 3 is amantadine concentration 267 μMs (reaching the standard grade), 2670 μMs (rolling off the production line) and rat hepatocytes hatch rear former medicine residual content in vitro altogether;
Wherein, × representing standard substance, ◇ represents gas phase sample;
Amantadine: amantadine; Amethopterin: methotrexate;
Acetaminophen: paracetamol; Quercetin: quercitrin (Huang) element;
Remaining: residual; Time: time;
Min: minute.
Fig. 4 is that 550 μMs of spermidines and rat hepatocytes hatch rear N-acetylize spermidine in vitro altogether;
Spermidine; Spermidine, spermidine; Amethopterin: methotrexate;
Acetaminophen: paracetamol; Quercetin: quercitrin (Huang) element;
As parent at T=0 accounts for the per-cent of female medicine zero time; The time time;
Min: minute.
Fig. 5 is that 550 μMs of spermidines and rat hepatocytes hatch rear former medicine residual content in vitro altogether;
Spermidine: spermidine, spermidine; Amethopterin: methotrexate;
Acetaminophen: paracetamol; Quercetin: quercitrin (Huang) rope;
Remaining: residual; Time: time;
Min: minute.
To be that 200 μMs of L-DOPA and rat hepatocytes are external hatch rear N-acetylize L-DOPA formation volume to Fig. 6 altogether;
L-Dopa: levodopa; Amethopterin methotrexate;
Quercetin: quercitrin (Huang) element; Time: time;
Asparent atT=0: the per-cent accounting for female medicine zero time; Min: minute.
To be that 200 μMs of L-DOPA and rat hepatocytes are external hatch rear former medicine residual quantity to Fig. 7 altogether;
L-Dopa: levodopa; Amethopterin: methotrexate;
Quercetin: quercitrin (Huang) element; Remaining: residual;
Time: time; Min: minute.
To be that 20 μMs of L-DOPA and rat hepatocytes are external hatch N-acetylize L-DOPA formation volume to Fig. 8 altogether;
L-Dopa: levodopa; Amethopterin methotrexate;
Quercetin: quercitrin (Huang) element; Time: time;
As parent at T=0 accounts for the per-cent of female medicine zero time; Min: minute.
Figure 92 0 μM of L-DOPA and rat hepatocytes is external hatches former medicine residual quantity altogether;
L-Dopa: levodopa; Amethopterin methotrexate;
Quercetin: quercitrin (Huang) element; Remaining: residual;
Time: time; Min: minute.
Embodiment
The present invention have studied a kind of medical detection technique, i.e. a kind of spermidine/spermine N 1-Transacetylase (is called for short: SSAT) active method, provides a kind of method that acetylize metabolite by detecting SSAT substrate analyzes SSAT activity, being convenient to the convenient of medical industry and safe handling.
The specific experiment process just implemented below is further elaborated and illustrates.
Specific experiment process of the present invention is as follows:
1, materials and methods:
Reagent
Methyl alcohol (EMD company, HPLC level); Dimethyl sulfoxide (DMSO) (is called for short: DMSO) (Sigma company, ACS); Distilled water (BRIVAL company, ASTM); Formic acid (Fisher Scientific company, ACS); Hydrochloric acid (Fisher Scientific company, ACS); Trypan blue solution (Sigma company, 0.4%, sterile filtration); Tetramethyl-azo azoles salt (Sigma company) Krebs-Henslaite damping fluid (AP Sciences company).
1.1 substrate
Table 2, acetylization reaction substrate
Remarks: be applied to this and study all concentration of substrate and revise according to indicated chemical purity.
1.2NAT1 and NAT2 pathway inhibitor
Table 3, NAT1 and NAT2 pathway inhibitor
Remarks: be applied to this and study all concentration of substrate and revise according to indicated chemical purity
1.3N-acetylize metabolite: following metabolite is as reference standard correction in this research LC/MS/MS bioanalysis
N-acetylize metabolite in table 4, LC/MS/MS bioanalysis
Remarks: be applied to this and study all concentration of substrate and revise according to indicated chemical purity.
Mark in 1.4
Mark in table 5, LC/MS/MS bioanalysis
Remarks: purity does not have correction calculation.
2, the former liver cell preparation of freezing female mouse
Adopt 20 rats (Wistar rat, female, 200 ~ 300g) liver cell, cell adopts Celsis ex vivo technique to obtain, stored under refrigeration in liquid nitrogen.Use cryopreserved hepatocytes thawed 37 DEG C of water-baths the day before yesterday, make suspension with substratum, centrifugal 10 minutes of 100x g, (be called for short: KHB) suspendible again, as liver cell storing solution with Krebs-Hensleite damping fluid.
Before induction experiment, hepatocellular concentration and active employing determination of trypan blue staining, in preliminary experiment, rat hepatocytes activity is 95% (Hu4161, Hu4198 and Hu4201).Be confirmed as 93% repeating to test (Hu4227) hepatocytes activity of using.By measuring hepatocyte activity, cell storing solution adds KHB and adjusts cell concn to target level 0.51x10 3/ ml, is equivalent to 2.5X10 5viable cell/0.49ml.
First rat hepatocytes was measured with trypan blue exclusion active before plating.
Prepared by table 6, substrate, inhibitor solution
3, solution preparation
(1) solubleness evaluation
37 DEG C add Krebs-Henslaite damping fluid, add or do not add 0.1 ~ 0.2%DMSO, measure its final concentration in substrate, the inhibitor aqueous solution.
Prepare each chemical storing solution, then use preheating KHB (37 DEG C) to be diluted to test fluid.Test fluid is prepared into 3 concentration, and HPLC/UV measures its optical density, makes typical curve, obtain linear regression relation with optical density-concentration
(2) mtt assay rat hepatocytes cytotoxicity detects
Mtt assay is adopted whether to have cytotoxicity to the former hepatocytes of refrigeration for detection substrate and inhibitor when detectable level.
Adopt 96 orifice plates, liver cell (0.5X106/ml) respectively with substrate, the material standed for (0,1/100,1/50 of different concns, 1/20,1/10,1/5,1/2, the higher target level of 1 times), add NAT1 or NAT2 or NAT1 and NAT2 simultaneously, hatch 15m altogether.Then preincubate 10 minutes, every hole adds the KHB (final concentration 0.5mg/ml) containing equivalent MTT, then hatches 30m.After hatching, remove substratum, add 100 μ l DMSO and dissolve, then use deck vibrator hydrotropy, measure the optical density of solution with microplate reader 540nm.
(3) rat hepatocytes N-acetylize is measured
Adopt 24 well culture plate vitro culture, incubator temperature 37 DEG C, containing 5%CO 2, 95% air.Each substrate or material standed for and NAT1 or NAT2 inhibitor are hatched separately or hatch altogether, parallel in duplicate.
Add certain density inhibitor or solvent control (5 μ L) to culture plate.Then each hole adds containing about 2.5X10 5active Hepatocyte storing solution 0.49ml, hatches 10min altogether.Then preincubation 10min, adds the mixture of each substrate of 5 μ L, material standed for or surrogate respectively, continues to hatch 0, and 30 and 60min.At the end of cultivation, in every hole, add 0.5ml ice methyl alcohol stopped reaction.
Due to solubleness restriction, L-DOPA is independently cultivated, instead of mixes with other four material standed fors.Parallel two aliquot sample in every hole, before LC/MS and LC/MS/MS analyzes potential N-acetylize metabolite, are stored in 1.5ml bottle, are kept at-80 DEG C (-72 DEG C ~-88 DEG C).
(4) LC/MS and LC/MS/MS method is to the bioanalysis of N-acetylize metabolite
This research adopts the N-acetylize metabolite of amantadine and sulphamethazine in LC/MS/MS quantitative analysis rat hepatocytes sample.N-acetylsulfanilamide diformazan pyrimidine and N-acetyl amantadine are respectively with N-acetylsulfanilamide diformazan pyrimidine-D 4with N-acetyl-D 3-amantadine is interior mark.Calibration standard: N-acetyl amantadine 0.1 ~ 200ng/ml, N-acetylsulfanilamide diformazan pyrimidine 1 ~ 2000ng/ml.QC sample concentration is respectively: N-acetyl amantadine 0.2,80 and 160ng/ml; N-acetylsulfanilamide diformazan pyrimidine 2800,1600ng/ml.
Due to this research carry out time, there is no available metabolite reference standard, thus with target spot metabolite know method for distinguishing monitored spermidine, para-aminosalicylic acid, para-amino benzoic acid, L-3,4 dihydroxyphenylalanine N-acetylize metabolite.Deriving from In Cultured Rat liver cell sample first for scanning medicine prototype, then carrying out the scanning of potential N-acetylize metabolite, the precursor of parent scanning characteristic fragment, carries out directed multiple-reaction monitoring simultaneously
4, results and analysis
(1) drug candidate, substrate and inhibitor are water-soluble
Water-soluble when liver cell incubation buffer reaches target final concentration of substrate when have evaluated 37 DEG C in KHB damping fluid, inhibitor.Dimethyl sulfoxide (DMSO), methyl alcohol or 0.5MHCl hydrotropy can be added when medicine is soluble in 0.1% ~ 0.2%KHB, assess solubility promoter by linear UV absorption process detection experiment concentration.Amantadine, spermidine are solvable.
(2) cytotoxicity of cryopreserved primary rat hepatocyte
Substrate adds or do not add NAT1, NAT1/NAT2 inhibitor does not have cytotoxicity to hepatocytes, except amantadine is at maximum concentration 2645 μMs (having cytotoxicity).Three inhibitor, only have (±) methotrexate, (±) methotrexate and acetparaminosalol phenol mixture, or Quercetin does not have cytotoxicity in test concentrations to rat hepatocytes yet.
(3) the acetylizad positive control of the N-of NAT1 and NAT2 in rat hepatocytes
NAT1 specific inhibitor Rheumatrex concentration 50 μMs, NAT2 specific inhibitor acetaminophen concentration 2144 μMs, at the end of hatching, N-acetylize is about 0.15%.
Inhibition NAT1 and NAT2 that be formed in of metabolite coexists suppressed 28% (Fig. 3 and table 7) of 60min.When there being NAT1 and NAT2 inhibitor to exist, 54%N-acetylize is suppressed.
(4) LC/MS/MS quantitative analysis N-acetyl-sulphamethazine and N-ethanoyl-amantadine
N-acetyl-sulphamethazine and ethanoyl-amantadine quantitative analysis adopt N-acetylsulfanilamide diformazan pyrimidine-D 4with N-acetyl-D 3-amantadine is interior mark.N-ethanoyl-amantadine correcting range 0.1 ~ 200ng/ml, N-acetyl-sulphamethazine correcting range 1 ~ 2000ng/ml.The matched curve of LC/MS/MS quantitative analysis correction is origin-excluded quadratic regression equation, and all concentration uses 1/x weighting factor.
N-acetyl-sulphamethazine:
The coefficient of determination: R^2=0.998133
Calibration curve :-1.4497e-007*x^2+0.00454808*x+6.02654e-005
N-ethanoyl-amantadine:
The coefficient of determination: R^2=0.999811
Calibration curve: 2.81933e-006*x^2+0.112015*x+0.00309319
The calibration standard curve of N-acetyl-sulphamethazine and N-ethanoyl-amantadine is Fig. 1, Fig. 2 respectively.
(5) the N-acetylize of rat hepatocytes SSAT positive control
SSAT substrate two positive controls are adopted: amantadine and spermidine in research.In rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor share, or the common specific inhibitor of NAT1 and NAT2, independently assess N-acetylizing.
NAT1 specific inhibitor methotrexate concentration is 50 μMs.NAT2 specific inhibitor acetaminophen limits due to solubleness, and concentration is 2144 μMs.The common inhibitor quercetin concentration of NAT1 and NAT2 is 480 μMs.In rat hepatocytes sample, N-acetylize metabolite derives from rat hepatocytes and amantadine (267 μMs and 2670 μMs) is hatched altogether, and N-acetyl metabolite amount is too low and could not detect.Concentrated rat hepatocytes and 267 μMs of amantadines are hatched sample and N-ethanoyl-amantadine still can not be detected.And amantadine residuum has almost no change (Fig. 3 and table 7) when hatching.Result is consistent with BRI No.BIM-2009-001 result of study.In rat urine, low-level N-acetylize-amantadine is checked through in vivo in research.
Former medicine residuum ratio is calculated by following formula:
The acetylized admantadine that table 7, different initial substrate concentration are formed and former medicine residual quantity
Note: lower limit of quantitation=0.1ng/mL
Detected result shows: the N-acetylize metabolite of spermidine, forms appearance fast at 0.At the end of hatching, quantity is close to 14% of the former medicine quantity added.This acetylization reaction does not suppress because of NAT1 inhibitor (50 μMs of methotrexates) and NAT2 inhibitor (2144 μMs of acetaminophen).But 26% acetylization reaction is suppressed when there is merely NAT1 inhibitor (50 μMs of methotrexates); use 480 μMs of Quercetins also can produce restraining effect, inhibitor acts on NAT1 and NAT2 two paths (Fig. 4, Fig. 5 and table 8).Former medicine is residual not to be changed.Meta-bolites, metabolism suppress, the remaining discordance of former medicine can be solved by quantitative analysis.
Table 8, rat hepatocytes and SSAT substrate 550 μMs of spermidines
Add or do not add NAT1 and NAT2 inhibitor is external hatch altogether after, N-acetylize metabolite and former medicine residual content
SSAT substrate positive control and NAT1 and NAT2 inhibitor test-results show SSAT substrate spermidine N-acetylize can occur in female rats hepatocyte model.The effect of N-ethanoyl is not combined suppression (methotrexate 50 μMs and acetaminophen 2144 μMs) by NAT1 inhibitor and NAT2 inhibitor.But application methotrexate 50 μMs or acetaminophen 2144 μMs but have impact separately.Quercetin adopts concentration 480 μMs, in so high concentration, non-specific retarding effect may occur.In this research, confirm that Optimized model parameter comprises inhibitor concentration further, be one of this project undeveloped reserve.
(6) acetylizing of rat hepatocytes substrate L-DOPA
SSAT substrate material standed for L-DOPA (20 μMs and 200 μMs) is adopted in research; in rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor share; or the common specific inhibitor of NAT1 and NAT2, independently assess N-acetylizing.
Result shows: acetylize metabolite increases along with incubation time extends generation.When the L-3,4 dihydroxyphenylalanine added is 200 μMs, produce 0.5% metabolite; When the L-3,4 dihydroxyphenylalanine added is 20 μMs, producing the metabolite of 5.2%, there is the saturated or self-retarding effect of reaction in prompting.When the L-3,4 dihydroxyphenylalanine added is 20 μMs, former medicine residual quantity is lower.There is the common inhibitor (Quercetin of NAT1 and NAT2,480 μMs) when existing, metabolite is formed significantly suppressed (> 90%), and NAT1 inhibitor (methotrexate, 50 μMs) and NAT2 inhibitor (acetaminophen, 2144 μMs) exist, metabolite forms not suppressed (Fig. 6,7,8,9, table 9).
Table 9, L-DOPA add or do not add NAT1 and NAT2 inhibitor is external hatch altogether after, N-acetylize metabolite and former medicine residual content
Remarks 1: suppose metabolite and former medicine plasma efficiency
(7) conclusion
Show from above result, can produce NAT1 and NAT2 substrate N-acetylizing in female rats hepatocyte model, application NAT1 inhibitor methotrexate and the acting in conjunction of NAT2 inhibitor acetaminophen can suppress N-acetylization reaction.On the contrary, there is the N-ethanoyl effect that can not change SSAT substrate spermidine in these two inhibitor, also demonstrate the specificity of these two NAT inhibitor simultaneously.
Result also shows L-3,4 dihydroxyphenylalanine and is better than NAT1 and NAT2 to SSAT selectivity, and the known undetected acetylizing of SSAT substrate amantadine may be because sample is different.SSAT raises and being proportionate of cancer, therefore the L-3,4 dihydroxyphenylalanine cancer markers that can regulate as SSAT.
In the present invention, the embodiment of above-described embodiment and the following stated is all to set forth the present invention better, is not for limiting the scope of the invention.Below by embodiment, the present invention is described in detail.
Embodiment 1,
Adopt 20 rats (Wistar rat, female, 200-300g) liver cell, cell adopts Celsis ex vivo technique to obtain, stored under refrigeration in liquid nitrogen.Use cryopreserved hepatocytes thawed 37 DEG C of water-baths the day before yesterday, make suspension with substratum, centrifugal 10 minutes of 100x g, with Krebs-Hensleite damping fluid (KHB) suspendible again.Adopt 24 orifice plate vitro culture, culture temperature 37 DEG C, containing 5%CO 2, 95% air, each hole adds containing about 2.5 × 10 5active Hepatocyte storing solution 0.49ml, hatches 10min altogether.Then preincubation 10min.In liver cell solution, one group adds L-3,4 dihydroxyphenylalanine is 200 μMs, inhibitor Quercetin 480 μMs, one group adds L-3,4 dihydroxyphenylalanine is 200 μMs, inhibitor methotrexate 50 μMs, one group adds L-3,4 dihydroxyphenylalanine is 200 μMs, inhibitor acetaminophen 2144 μMs, the mixing solutions 5 μ l of each substrate, inhibition is to culture plate, continue to hatch 0,30 and 60min.At the end of cultivation, in every hole, add 0.5ml ice methyl alcohol stopped reaction.Often group is duplicate.Measurement result is in table 10.
Table 10,200 μMs of L-DOPA add or do not add NAT1 and NAT2 inhibitor is external hatch altogether after N-acetylize metabolite and former medicine residual content
Embodiment 2,
Adopt 20 rats (Wistar rat, female, 200 ~ 300g) liver cell, cell adopts Celsis ex vivo technique to obtain, stored under refrigeration in liquid nitrogen.Use cryopreserved hepatocytes thawed 37 DEG C of water-baths the day before yesterday, make suspension with substratum, centrifugal 10 minutes of 100x g, with Krebs-Hensleite damping fluid (KHB) suspendible again.Adopt 24 orifice plate vitro culture, culture temperature 37 DEG C, containing 5%CO 2, 95% air, each hole adds containing about 2.5 × 10 5active Hepatocyte storing solution 0.49ml, hatches 10min altogether.Then preincubation 10min.In liver cell solution, one group adds L-3,4 dihydroxyphenylalanine is 20 μMs, inhibitor Quercetin 480 μMs, one group adds L-3,4 dihydroxyphenylalanine is 200 μMs, inhibitor methotrexate 50 μMs, one group adds L-3,4 dihydroxyphenylalanine is 200 μMs, inhibitor acetaminophen 2144 μMs, the mixing solutions 5 μ l of each substrate, inhibition is to culture plate, continue to hatch 0,30 and 60min.At the end of cultivation, in every hole, add 0.5ml ice methyl alcohol stopped reaction.Often group is duplicate.Measurement result is in table 11.
Table 11,20 μMs of L-DOPA add or do not add NAT1 and NAT2 inhibitor is external hatch altogether after N-acetylize metabolite and former medicine residual content

Claims (13)

1. for detecting a method for the acetylize metabolite of SSAT substrate, it is characterized in that, described method comprises the following steps:
1) hatch a certain amount of a kind of medicine, described medicine and SSAT substrate are L-3,4 dihydroxyphenylalanines, and described hatching refers to is hatched altogether by medicine and mammalian tissues or cell;
2) certain cell analysis sample is obtained;
3) the acetylize metabolite in analytic sample is detected;
Described method mainly or only regulates the acetylizad specific drugs material standed for of N-by SSAT for assessment of in potential SSAT path.
2. the method for the acetylize metabolite for detecting SSAT substrate according to claim 1, is characterized in that, described Mammals comprises people or other animals.
3. the method for the acetylize metabolite for detecting SSAT substrate according to claim 2; it is characterized in that, other described animals comprise in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or rhesus monkey one or more.
4. the method for the acetylize metabolite for detecting SSAT substrate according to claim 3, is characterized in that, other described animals are one or more in mouse, rat, monkey, pig, rabbit or dog.
5. the method for the acetylize metabolite for detecting SSAT substrate according to claim 4, is characterized in that, other described animals are one or more in rat or monkey.
6. the method for the acetylize metabolite for detecting SSAT substrate according to claim 1, is characterized in that, the dosage range of described medicine is 1 ~ 10mg/kg.
7. the method for the acetylize metabolite for detecting SSAT substrate according to claim 6, is characterized in that, the dosage range of described medicine is 3 ~ 6mg/kg.
8. the method for the acetylize metabolite for detecting SSAT substrate according to claim 1, is characterized in that, described analytic sample derives from the mammiferous cell sample that 0 ~ 24h hatches altogether.
9. the method for the acetylize metabolite for detecting SSAT substrate according to claim 8, is characterized in that, described analytic sample derives from the mammiferous cell sample that 10 ~ 60min hatches altogether.
10. the method for the acetylize metabolite for detecting SSAT substrate according to claim 9, is characterized in that, described analytic sample derives from the rat hepatocytes that 10 ~ 60min hatches altogether.
The method of the 11. acetylize metabolites for detecting SSAT substrate according to claim 1; it is characterized in that; acetylize metabolite method in described detection analytic sample is the measured quantity according to acetylize metabolite in Mammalian samples, makes typical curve to determine the activity of SSAT in mammalian body.
The method of the 12. acetylize metabolites for detecting SSAT substrate according to claim 11; it is characterized in that, in described Mammalian samples the determination techniques of acetylize metabolite content be comprise in chromatographic detection method, radio-labeling, mass spectrum, infrared, near infrared or ultraviolet detection one or more.
The method of the 13. acetylize metabolites for detecting SSAT substrate according to claim 12; it is characterized in that, described chromatographic detection method be comprise in gas-chromatography, high performance liquid chromatography, thin-layer chromatography, specific antibody chromatogram or antibody affinity chromatography one or more.
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