CN102344950A

CN102344950A - Method for detecting activity of spermidine/spermine N1-acetyltransferase

Info

Publication number: CN102344950A
Application number: CN201110145069XA
Authority: CN
Inventors: 郑启明; 阿曼得·拉希德; 李铁军; 周辉
Original assignee: Biomark(China) International Ltd
Current assignee: Biomark(China) International Ltd
Priority date: 2011-05-31
Filing date: 2011-05-31
Publication date: 2012-02-08
Anticipated expiration: 2031-05-31
Also published as: CN102344950B

Abstract

The invention, relating to the technical field of medicine, relates to a method for detecting the activity of spermidine/spermine N1-acetyltransferase, that is, a quantitative technique of analyzing SSAT activity through detecting acetylated metabolites of the SSAT substrate. According to the invention, the N-acetylation is regulated mainly through or only through SSAT in SSAT pathway and the specific substrate in the N-acetylation regulation is L-dopa, partial metabolism of the substrate can generate an acetylated metabolite N-acetyl-L-dopa thought the introduction of SSAT, the SSAT upregulation is positively correlated with cancer, accordingly, L-dopa can be used as a cancer mark regulated by the SSAT upregulation, and a novel detection method is provided for early detection, early diagnosis, early treatment and the like of tumors. The method has the advantages of safely, efficiency, strong practicality, convenient and fast operating method, simple usage, high positive incidence, and remarkable effect, and can be used as an auxiliary means for preventing, diagnosing, detecting, protecting, treating and studying various tumors.

Description

A kind ofly be used to detect spermidine/spermine N 1The method of-acetyltransferase activity

Technical field

The present invention relates to medical technical field, specifically relate to a kind of medical detection technique, more particularly relate to a kind of spermidine/spermine N ¹-Transacetylase (is called for short: SSAT) active method; Specifically relate to a kind of acetylize metabolite again and analyze the active method of SSAT through detection SSAT substrate; Wherein: the SSAT substrate is a levodopa, and the part metabolism of levodopa is can produce acetylize metabolite N-ethanoyl-L-DOPA through inducing of SSAT.

Background technology

(1) research overview of levodopa

1, general introduction

3-hydroxyl-L-tyrosine/L-β-(3; The 4-dihydroxy-benzene) L-Ala/L-3-(3; The 4-dihydroxy-benzene) L-Ala/L-(3; The 4-dihydroxy-benzene) L-Ala (Levodopa); Claim that again the L-DOPA (is called for short: L-Dopa; L-DOPA), being the hydroxylate of tyrosine, is the intermediate product of the synthetic catecholamine of L-tyrosine 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.

Attach: the left-handed enantiomorph that is meant organic compound with dextrorotation is to making light to inhour or clockwise direction rotation respectively in the polarized light.Can make the left-handed or dextral isomer of polarized light can be called as levo form and dextrorotatory form.In organic chemistry, left-handed with (+) expression usually, (-) expression dextrorotation.Like the levotartaric acid of L-configuration, its full name can be write L (+)-tartrate.

L-DOPA is an antiparkinsonian drug, is applicable to primary parkinsonism and non-medicine originality Parkinsonism.L-DOPA gets into cerebral tissue through hemato encephalic barrier, plays a role.Untoward reactions such as nauseating, vomiting, poor appetite and insomnia, illusion after taking, L-DOPA are arranged.

2, physico-chemical property

English another name: 3-(3,4-Dihydroxyphenyl)-L-alanine, L-3-(3,4-Dihydroxyphenyl) alanine

Chemical name: 3-hydroxyl-L-tyrosine

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-11.7º (c=5.3,1N HCl)

Fusing point: 295 ℃

3, pharmaceutical analysis

L-DOPA production has chemosynthesis, extracted form natural plant and three main paties of microbial enzyme conversion method; 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 in mammalian body with in the brain, be synthesized by L-tyrosine.

(1) method name: the mensuration-nonaqueous titrations of levodopa bulk drug-levodopa.

Range of application: present method adopts the content of levodopa in the titration measuring levodopa bulk drug.

Present method is applicable to the levodopa bulk drug.

Method principle: after trial-product adds the anhydrous formic acid dissolving, add Glacial acetic acid and shake up, add 2 of Viola crystallina indicating liquids; Show green with the perchloric acid titration drop is fixed to solution; And titrating result proofreaied and correct with blank test, according to the 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.

Specimen preparation:

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, put coldly, add anhydrous Glacial acetic acid and make into 1000mL in right amount, shake up, placed 24 hours.If the easy acetylize of institute's trial-product of surveying, then must measure the water content of this liquid with aquametry, to be adjusted to the water content of this liquid be 0.01%～0.2% for water and aceticanhydride again.

Demarcation: be taken at 105 ℃ of about 0.16g of benchmark Potassium Hydrogen Phthalate that are dried to constant weight, the accurate title, decide, and adds anhydrous Glacial acetic acid 20mL and make dissolving, adds 1 of Viola crystallina indicating liquid, and slowly titration is extremely blue with this liquid, and titration results is proofreaied and correct with blank test.Every 1mL perchloric acid titration liquid (0.1mol/L) is equivalent to the Potassium Hydrogen Phthalate of 20.42mg.According to the amount of taking of the consumption and the Potassium Hydrogen Phthalate of this liquid, 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 by weighing the about 0.1g of trial-product, adds anhydrous formic acid 2mL and makes dissolving, adds Glacial acetic acid 20mL and shakes up, and adds 2 of Viola crystallina indicating liquids, shows green with perchloric acid titration liquid (0.1mol/L) titration to solution, and titrating result is proofreaied and correct with blank test.Every 1mL perchloric acid titration liquid (0.1mol/L) is equivalent to the C9H11NO4 of 19.72mg.

Annotate: " precision takes by weighing " mean take by weighing weight should be accurately the thousandth of the weight that takes by weighing extremely." precision is measured " means that the accuracy of measuring volume answers in the National standard accuracy requirement to this volume transfer pipet.

Reference: Pharmacopoeia of People's Republic of China, Chinese Pharmacopoeia Commission compiles, Chemical Industry Press, version in 2005, one one, p.85.

(2) method name: the mensuration-spectrophotometry of levodopa capsule-levodopa

Range of application: present method adopts the content of levodopa in the spectrophotometry levodopa capsule.

Present method is applicable to the levodopa capsule.

The method principle: trial-product adds hydrochloric acid soln and processes test liquid, puts ultraviolet-visible pectrophotometer, measures optical density in the 280nm wavelength, calculates its content.

Reagent: hydrochloric acid soln

Plant and instrument: ultraviolet-visible pectrophotometer

Specimen preparation:

1. hydrochloric acid soln is got hydrochloric acid 9mL, adds water and makes into 1000mL in right amount, shakes up.

2. the content under the content uniformity item is got in the preparation of need testing solution, mixes, and precision takes by weighing in right amount (being equivalent to levodopa 30mg approximately); Put in the 100mL measuring bottle; It is an amount of to add hydrochloric acid soln, and jolting makes the levodopa dissolving, is diluted to scale with hydrochloric acid soln; Shake up; Filter, precision is measured subsequent filtrate 10mL and is put in another 100mL measuring bottle, adds hydrochloric acid soln and is diluted to scale; Shake up, promptly get need testing solution.

Annotate: " precision takes by weighing " mean take by weighing weight should be accurately to the thousandth that takes by weighing weight." precision is measured " means that the accuracy of measuring volume answers in the National standard accuracy requirement to this volume transfer pipet.

Operation steps: getting need testing solution according to ultraviolet spectrophotometry, measure optical density in wavelength 280nm place, is 141 calculating by the uptake factor (E1%1cm) of C9H11NO4, promptly gets.

Annotate: spectrophotometry should be contrast with same batch of solvent of preparation trial-product, adopts the quartzy cuvette of 1cm.With the maximum wavelength of optical density as measuring wavelength, the optical density reading of general trial-product, 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 that records is on the low side.The selection of slit width, should be when reducing slit width the optical density of trial-product no longer increase and be as the criterion.Because cuvette and solvent itself have blank the absorption, so should deduct blank reading after measuring the optical density of trial-product, calculate content again.

Reference: Pharmacopoeia of People's Republic of China, Chinese Pharmacopoeia Commission compiles, Chemical Industry Press, version in 2005, one one, p.86.

4, pharmacological toxicology

These article are for intending the Dopaminergics antiparkinsonism drug; Levodopa is a synthetic dopamine precursor material in the body, and itself is parmacodynamics-less activity also, gets into maincenter through hemato encephalic barrier; Change into Dopamine HCL and bring into play pharmacological action through the dopa decarboxylase effect, improve the Parkinson's disease symptom.Because these article can increase neurotransmitter such as Dopamine HCL and norepinephrine in the brain, can also improve the tolerance of brain to ammonia, and be used to treat hepatic coma, improve the maincenter function, make patient clear-headed, doing well,improving.

(1) the anti-parkinson levodopa changes Dopamine HCL in brain, replenishes insufficient dopamine in the striatum, thereby has the curative effect of anti-parkinson.Research shows, once uses the patient of a large amount of levodopa treatment, after death in the striatum dopamine concentration than the medication curer is not high 5～8 times; And dopamine concentration is consistent with the curative effect of levodopa in the brain.Illustrate that remaining dopaminergic neuron still has the ability that stores Dopamine HCL in patient's black substance-striatum path.Its striatum dopa decarboxylase still has enough enzymic activitys can make levodopa change Dopamine HCL into.

After levodopa treatment, about 75% patient obtains better curative effect.Treatment initial stage curative effect is more apparent.The effect characteristics of levodopa are:

1. to light disease and better than the young patient curative effect, and severe and old weak patient's weak curative effect;

2. better to muscular rigidity and dyskinesia curative effect, and to muscular tremor symptom weak curative effect, still can take effect to the latter like long-term prescription and than heavy dose of;

3. effect is slower, often needs medication 2～3 week the improvement of objective sign just to occur, just obtains greatest treatment efficacy more than 1～6 month, but persistent, and increases progressively with the administration time prolongation.

Levodopa is also effective to the parkinson's syndrome that other reasons causes.But caused invalid to antipsychotic drugs such as phenothiazines, the effect of blocking-up central dopaminergic receptor is arranged because of these medicines.

(2) the pseudo-mediator theory that is right of treatment hepatic coma hepatic coma pathogenesis is thought all oxidized detoxifcations in liver of normal body protein metabolism product phenylethylamine and tyrasamine.Liver dysfunction, blood phenylethylamine and tyramine increase in nerve cell Neijing β-hydroxylase respectively generate pseudo neurotransmitters, phenylethanolamine and hydroxyl phenylethanolamine (octopus amines), which replaces the normal neurotransmitter - norepinephrine, impede nerve function.Can in brain, change norepinephrine with levodopa.Make normal nervous activity be able to recover, the patient can be transferred to by stupor and reviving.Because of not improving liver function, act on just temporary.

5, drug use

L-DOPA is an antiparkinsonian drug.Get into cerebral tissue through hemato encephalic barrier, be transformed into Dopamine HCL, play a role through the dopa decarboxylase decarboxylation.

L-DOPA is used for primary parkinsonism and non-medicine originality Parkinsonism, and centering, slight effect are better, and severe or the elderly are relatively poor.

(1) usage and consumption

Preparation specification: tablet or capsule: 0.25g.

Oral: Parkinsonism, begin 0.25～0.5g/ time, 3～4 times/day, whenever increased by 0.125～0.5g at a distance from 3～4 days.Maintenance dose 3～6g/ day, divide clothes 3～4 times, in the dosage escalation process as occur to feel sick etc., should stop increment, treat to increase again behind the transference cure.As with the same usefulness of dopa decarboxylase inhibitor, dosage can reduce by 50%.

(2) pharmacokinetics

L-DOPA is a kind of important biological material in the organism; Be catecholamines neurotransmitter dopamine, norepinephrine or adrenergic precursor; From L-tyrosine to catechol or the important intermediate the melanic biochemical metabolism approach process; Can directly (be called for short: COMT) change into the 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 the healthy person body, is very important but be 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 following:

Relevant English in the above-mentioned pathways metabolism is explained as follows:

L-dopa: L-dopa;

S-adenosyl-L-metthionine:s-adenosine-L-methionine(Met);

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;

The 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.

The oral back of L-DOPA is by little intestinal absorption.Behind the oral levodopa; Active transport system through die aromatischen Aminosaeuren absorbs rapidly from small intestine; About 0.5～2 hour; The Plasma Concentration peaking; Be distributed widely in each tissue in the body; 1% entering maincenter changes into Dopamine HCL and plays a role, all the other major parts all outside brain the metabolism decarboxylation become Dopamine HCL, so onset is slow.

The oral back 80% of L-DOPA was degraded into the Dopamine HCL metabolite in 24 hours, be mainly homovanillic acid and dihydroxyphenyl acetic acid, by renal excretion; The small portion levodopa changes melanochrome (melannin) into; Some levodopa (is called for short: COMT) methylate, change 3-methoxyl group DOPA into through catechol-O-methyl transferase (COMT) in addition; Above metabolite is drained rapidly by kidney.This metabolic process consumes more COMT, and methyl can cause methionine deficiency mainly from the methionine(Met) in the food so take levodopa for a long time in the COMT reaction.

Some metabolite can make urinates the look that reddens.It is about 5% that prototype excretes, can be through lactation.

Its uptake rate is subjected to multiple factor affecting, delays have other amino acid to compete active transport system (like high protein diet) etc. with it in (with clothes cholinocepter blocking agent), gastric acidity height or the small intestine like stomach emptying, all can reduce its bioavailability.After the absorption, major part is transformed into Dopamine HCL by decarboxylation during first through liver.Also there is considerable part in intestines, the heart, kidney, to be generated Dopamine HCL by decarboxylation.And Dopamine HCL is difficult for seeing through hemato encephalic barrier, and therefore the levodopa that gets into central nervous system is less than 1% of consumption.In peripheral tissues, forming a large amount of Dopamine HCLs is the reasons that cause untoward reaction.

Plasma half-life (t _1/2) be 1～3 hour, as taking periphery dopa decarboxylase inhibitor (carbidopa) simultaneously, can reduce the consumption of levodopa, the amount that makes it to get in the brain increases, and can reduce the untoward reaction that the periphery Dopamine HCL causes.

(3) untoward reaction

Gastrointestinal reaction is shown in the treatment initial stage as feeling sick, vomit, losing the appetite, and uneasiness, insomnia, illusion mental symptom can appear in medication after 3 months, still postural hypotension irregular pulse and involuntary movement etc. can be arranged in addition.

Untoward reaction is more.Be divided into early stage and long-term two big types.

1. digestive tube: the early stage gastrointestinal reaction incidence of the independent medication of these article is higher; Apocleisis, feel sick, vomiting, poor appetite; Abdominal discomfort, flatulence, dry, dysphagia, polysialia, tongue burning sensation, bitter taste, diarrhoea or constipation etc. worsen peptide ulceration, cause bleeding.Along with the prolongation of administration time, GI adverse reaction rate reduces, but still has groups of people can not tolerate produce effects dosage.As share with decarboxylase inhibitor, then GI tolerance is better, especially with food with clothes, gastral disorder is slight and be one to cross property, only has a little patient to add and uses antiemetic.

2. cardiovascular systems: orthostatic hypotension, palpitaition, tachycardia and hypertension.Also arrhythmia appears in some patient, mainly is because newborn Dopamine HCL acts on the cause of heart beta receptor.

3. central nervous system: grit one's teeth in tarantism appearance and involuntary action, hand tremor increase, bradykinesia outbreak, the sleep, ataxia, jerk, numbness, weakness, tired, headache, opisthotonus, entanglement, excitement, anxiety, glad, insomnia, nightmare, convulsions etc., phrenoplegia also has vain hope or illusion, major depression (comprising introgression) and hypomania.

4. blood system: hemolytic anemia, granulocytosis, oxyphorase and hematocrit value descend, oligoleukocythemia etc.

5. the eye: blepharospasm, diplopia, blurring of vision, platycoria etc.

6. other: have a running nose, bleed after flush, fash, urine and sweat blackout, many, the uroschesis of perspiring or incontinence, the menstruation, oedema, the storehouse coomb's test Coomb positive; The rising of of short duration alkaline phosphatase, Aspartic Acid transaminase, pyruvate aminotransferase, serum lactic dehydrogenase, bilirubin and blood urea nitrogen can be arranged; Urine protein, uric acid increase etc., idol can increase the weight of gout.But sexual function improving also.

6, interact

But the 1. Antiparkinsonian effect of benzene phenodiazine Bi class medicine and these article of phenytoin Sodium antagonism should be noted with the time spent.

But the 2. antidetonation of methyldopa or these article of the clonidine antagonism numbness effect of quivering increases the weight of the side effect of its postural hypotension.These article should not with the two same usefulness, also can not with the general amine of phenothiazines and methoxy-DDT fiber crops with using.

3. oxidase inhibitor can stop the degraded of Dopamine HCL in vivo, strengthens its effect, but can cause heart rate to be accelerated and hypertensive crisis, should not use together with these article.In the oxidase inhibitor of stopping using with these article first two weeks, or these article of stopping using used oxidase inhibitor after 2 days.

4. should not use together with serpentine and adrenomimetic drug class medicine.Serpentine can reduce the effect of these article, and adrenomimetic drug class medicine can increase the weight of cardiovascular untoward reaction.

5. GENERAL ANESTHETICS and these article share, and cardiovascular accident be prone to take place, should be before general anesthesia at least 1 day these article of stopping using.

6. vitamins B ₆For Dopamine HCL decarboxylase prothetic group, can increase peripheral tissues's decarboxylase, heavy dose of application can reduce the effect of these article.Unsuitable and the vitamins B of these article ₆With using.But as share vitamins B then with decarboxylase inhibitor ₆Can in brain, promote the Dopamine HCL decarboxylation, strengthen the effect of levodopa.

7. these article can be strengthened the antihypertensive effect of guanethidine and methyldopa, and they also can influence the Antiparkinsonian effect of these article simultaneously.

8. the extrapyramidal symptoms that causes of antipsychotic drug, unavailable article treatment is because of both have antagonistic action.

9. with carbidopa, Benserazide, Symmetrel, kemadrin with synergy is arranged, can reduce consumption and alleviate untoward reaction.

(α-methyldopahydrazine) has two kinds of isomer to MK-485; Its levo form claims that carbidopa (carbidopa) is stronger L-aromatic amino acid decarboxylase suppressor factor; Owing to be difficult for passing through hemato encephalic barrier; So when share with levodopa; The activity that only can suppress the periphery dopa decarboxylase; Thereby reduce the generation of Dopamine HCL, improve the concentration of Dopamine HCL in the brain simultaneously in peripheral tissues.Like this, can improve the curative effect of levodopa, can alleviate the side effect of its periphery again, so be the important adjuvant of levodopa.Carbidopa is used does not separately have pharmacological action basically.Carbidopa and the levodopa dosage by 1: 10 is share; Can make the effective dose of levodopa reduce 75%; Benserazide (benserazide) and carbidopa have same effect, and it and levodopa are claimed madopar (madopar) by the compound preparation of processing at 1: 4, are applied to clinical.

Amantadine (amantadine) is former to be antiviral drug, and the back finds that it also has the effect of anti-parkinson, the too late levodopa of curative effect, but be better than the cholinocepter blocking agent.It is short that instant effect and holding is imitated, and medication a couple of days can be obtained greatest treatment efficacy, but 6～8 week of logotype the back curative effects weaken gradually.Share synergy with levodopa.The mechanism of its anti-parkinson possibly be to impel complete dopaminergic neuron remaining in the striatum to discharge Dopamine HCL; And can suppress the re-uptake of Dopamine HCL; And effect and more weak cholinolytic effect that direct exciting Dopamine Receptors is arranged.Behind the long-term prescription, livedo reticularis appears in common skin of lower extremity, possibly be by due to the contraction of catecholamine release causing peripheral blood vessel.Idol causes convulsions, so epileptic's forbidding.Every day, dosage surpassed 300mg, but induced insomnia, spiritual uneasiness and ataxia etc.

10. Propranololum can be strengthened these article curative effect, also strengthens these article and induces the effect of stimulating growth hormone secretion.

7, precaution

Lighter patient of age is prone to " switch " phenomenon (the suddenly many moving uneasinesses of patient be can not be to be " pass " for the myotony motion then appears again in " opening "); Can reduce the upset of dosage or quiet notes levodopa or control this phenomenon; Share curative effect with vitamin B6 or chlorpromazine etc. reduces; Share the increase curative effect with periphery dopa decarboxylase inhibitor carbidopa etc.; Reduce side effect, can merge application vitamin B6 taboo this moment and oxidase inhibitor, ephedrine, serpentine and adrenomimetic drug share.Peptide ulceration, hypertension, psychosis, diabetes, irregular pulse and angle closure glaucoma patient forbidding.

1. forbid in irritated to these article, serious cardiovascular is sick, encephalosis, endocrine disturbance, psychosis, serious neurasthenia, used the agent of monoamine oxydasis, narrow-angle glaucoma, peptide ulceration in 2 weeks, convulsions history patient is arranged, and the early stage pregnant woman of gestation, wet nurse, puerpera and the children below 12 years old.

2. hemolytic anemia, liver or ephrosis, scheming infarct history, Peptic Ulcers, epilepsy, mental anomaly, tuberculosis, bronchial asthma and the careful usefulness of patient with gout are arranged.

3. these article as take on the feed the time, particularly high protein diet, but interference medicament is by the transfer of blood plasma to nervus centralis.Medication and advance to hang down the fluctuation that the albumen fast food can reduce clinical response between two meals.

4. because the degree of safety of these article is very little, should strictly grasp indication, detailed medical history-taking is also done inspection.Dosage is decided according to patient's tolerance, begins from low dose, increases gradually, and occurring until toxic reaction is that decrement is kept.The toxicity that occurs should in time be handled.

5. early treatment often has asymptomatic orthostatic hypotension; Also the someone occurs dizzy or faints, and continues medication and can take a turn for the better, and can tolerate after the general several months.During the medication, should be slow when movable or change position.

6. should note intraocular pressure, routine blood test and have or not anaphylaxis; The sugar of should often having a blood test.

7. these article generally show when 2～3 weeks the effect of Parkinsonism.Shake the quite aggravation of property paralysis period, continue 1～3 hour outbreak in 1 minute, and show effect at one time every day, the expression consumption is too big or be subjected to due to the emotional shock.

8. when the abnormal movement of involuntary muscle appears in the patient, decrement immediately, otherwise symptom will be aggravated.

9. during the medication, should avoid the edible B that is rich in as far as possible ₆Food, like yeast, whole wheat, wheat bran, internal organ and lean meat, vegetables with green leaves; Be not engaged in the dangerous activity of band, as driving, ascend a height etc.; When being in a bad mood, during the change of aspect such as intelligence, behavior, answering decrement.

10. during the medication, false uric acid rising in the false glucose in urine positive, the urine ketoboidies positive and the urine can be arranged.

8, clinical study

[effect cures mainly] carbidopa and levodopa share the treatment paralysis agitans.

The composite sheet of [chemical ingredients] new drug " carbidopa sheet (small pieces) " and old medicine " levodopa sheet (sheet) " combination packaging, every of small pieces contain carbidopa 25mg, and large stretch of every contains levodopa 0.25g.

The levodopa decarboxylation that [pharmacological action] kappa DOPA can suppress periphery becomes Dopamine HCL; It can not see through hemato encephalic barrier; Thereby make the levodopa that gets into low dosage in the brain be subjected to decarboxylation, reach the Dopamine HCL of effective concentration, and reduce the side effect of periphery Dopamine HCL.

[drug interaction] share with oxidase inhibitor can cause hypertension.

With vitamins B ₆, chlorpromazine or Papaverine share and can reduce the effect of these article.

Same with o-methyl-diphenhydramine with strengthening the effect of these article.

Vitamins B ₆Week is metabolized to Dopamine HCL outside can to quicken levodopa.

These article share with tricyclic antidepressant possibly produce hypertension and dyskinesia, should pay attention to.

After thiodiphenylamine, butylbenzene mountain amine, phenytoin Sodium and Papaverine and levodopa merge application, the report that has the levodopa curative effect to reduce.

[untoward reaction] carbidopa does not have special untoward reaction; Though it can reduce single adverse reaction rate that produces with levodopa; But when taking carbidopa and levodopa, dry, constipation still can occur, feel sick, untoward reactions such as vomiting, dizzy, many moving, palpitaition, hydrostomia.And the maincenter untoward reaction that these article can not alleviate levodopa takes place, like involuntary movement, dyskinesia, mental anomaly etc.

[contraindication] following patient's forbidding carbidopa and levodopa sheet: the incompetent patient of metabolism, the patient of sympathomimetic amine, narrow angle glaucoma patients such as cardiovascular, internal secretion, blood, liver, lung and kidney.Lactating women, the careful usefulness of pregnant woman.

[product specification] be levodopa 200mg 1., carbidopa 50mg; 2. levodopa 100mg, carbidopa 25mg.

[usage and dosage] is oral.When usual amounts begins one day with carbidopa 25～50mg, levodopa 0.25～0.5g, (one with 1～2 little lattice dress); After treating a week, every clothes 3～4 days 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) divides and takes for 3～4 times.The patient of single clothes levodopa sheet; As taking the carbidopa and levodopa sheet; The levodopa sheet should be stopped to obey and just these article can be taken later at least 8 hours; When taking; The predose of its levodopa should be equivalent to former single with 25% of dosage; Then more gradually dosage maximal dose every day carbidopa must not surpass 0.2g, levodopa must not surpass 2g (promptly must not surpass 8 little lattice dresses).

[storage practice] shading, airtight preservation.

[precaution] are single invalid with carbidopa, must share with levodopa.Use oxidase inhibitor person simultaneously, begin to stop using, should not use when outside medicine causes cone, reacting with fortnight before these article.

(2) spermidine/spermine N ¹The research overview of-Transacetylase

Cohen and Morgan were at report in 1998: SSAT extensively is present in the mammalian tissues, eliminates spermine at endocellular metabolism, however in inductive mammalian tissues normally or not SSAT be in extremely low-level.SSAT is the catabolic rate-limiting enzyme of polyamines, and the reverse of catalysis spermine turns to spermidine and the spermidine reverse turns to putrescine, can promote polyamines degraded, drainage, circulation and/or cell internal recycle.

Casero Pegg can induce generation by multiple medicine, somatomedin, polyamines, polyamine analogs, toxicant, hormone and physiological stimulation at report SSAT in 1993, but induces the period of generation different for each individuality.The expression regulation of SSAT can occur in and transcribe, mRNA stable, mRNA translates and the level of protein stabilization.

The SSAT gene comprises polyamines at transcription initiation site 1522-1492 and replys composition, and in these 31 pairs of basic sequences, the polyamines reaction is confirmed as 9 pairs of basic sequences.By polyamine analogs N ¹, N ¹²The SSAT of-two (ethyl) spermine or the mediation of natural polyamines inductive polyamines reacted constituent transcribes.

The SSAT molecular weight is less than 1000 in the rat hepatocytes; Be equivalent to by 60 of inducing cell; 000 molecule induces mammalian tissues can cause the SSAT level to improve by different inductors, thereby can be used as main (former) amine-containing compound potential acetylize individuality.

SSAT induce or normally there is competent SSAT enzyme in overexpression in cell, perhaps in experiment in vitro, use 100,000Xg centrifugal supernatant liquor can reach (Casero & Pegg 1993; Fogel-Petrovic 1997; Matsui & Pegg 1980; Pietila 1997).

SSAT is a kind of promotion substrate acetylase, particularly acetyl spermine and spermidine or its analogue.SSAT active level method depends on this area hi-tech personnel and complicated experimental technique at present.More particularly, need a kind of passing through that spermidine/spermine substrate acetylated form is detected the active quantivative approach of SSAT, comprise, detect separate pathological conditions thereby can be used as through detecting amantadine and L-DOPA.

In a word, through existing technical information analysis revealeds such as document retrievals, up to the present, find to have the report of aspects such as acetylize measuring method of activity, enzyme kinetics and SSAT spermidine and the spermine substrate of SSAT as yet.

Summary of the invention

The technical problem that will solve required for the present invention is to disclose a kind of detection Mammals spermidine/spermine N ¹The method of-acetyltransferase activity is promptly assessed in the potential SSAT path and mainly or is only regulated the acetylizad specific substrate of N-through SSAT, to overcome the above-mentioned defective that prior art exists.

That is to say that the present invention is through research and theory studies such as experimentation on animals, clinical trials, one of purpose is intended to provide a kind of and new spermidine/spermine substrate acetylated form is detected the active quantitative technique of SSAT.

Two of the object of the invention is mainly or only to regulate the acetylizad specific drugs material standed for of N-through SSAT in the assessment potential SSAT path.

(1) technical conceive

The contriver through the latest find of research is: L-DOPA is a kind of important biological material in the organism; Be catecholamines neurotransmitter dopamine, norepinephrine or adrenergic precursor; From L-tyrosine to catechol or the important intermediate the melanic biochemical metabolism approach process; Can directly (be called for short: COMT) change into the 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 the healthy person body, is very important but be 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 is can produce acetylize metabolite N-ethanoyl-L-DOPA through inducing of SSAT; Acetylize metabolite N-ethanoyl-L-DOPA through detecting SSAT substrate levodopa can be analyzed the SSAT activity; And being proportionate of active rise of SSAT and cancer; Therefore; The L-DOPA can be used as SSAT and raises the cancer markers of being regulated, with this index as early prevention, diagnosis, detection, protection, treatment and research cancer in medical treatment and the scientific research.

Further research detects the active quantitative technique of SSAT to spermidine/spermine substrate acetylated form; Improve the medical diagnosis quality and the scientific research effect of cancer; Realize discovery early, diagnosis morning, early treatment, avoid and alleviate the burden of family and society, help improving patient's life quality.

The patient that disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase lack particularly disturbances in patients with Parkinson disease is more and more; Have a strong impact on the health and the life quality of Chinese population; Bring white elephant for family and society; Develop the product that prevents, diagnoses, detects, protects, treats and study the aspects such as patient that help disturbances in patients with Parkinson disease or L-aromatic amino acid decarboxylase shortage; Particularly medicine, diagnostic reagent and healthcare products etc. have remarkable social benefit, economic benefit.

According to this idea and thinking, the contriver passes through experimental study and analysis repeatedly, successfully obtains the result of study and the application product of expecting.

(2) be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity

The present invention provides a kind of detection Mammals spermidine/spermine N ¹The method of-acetyltransferase activity mainly or is only regulated the acetylizad specific substrate of N-through SSAT in the assessment potential SSAT path.

The present invention regulates the acetylizad specific substrate of N-to SSAT and has carried out many-sided test and research at aspects such as prevention, diagnosis, detection, protection, treatment and researchs.According to the invention and the data description of reference in vivo, measure the active analytical procedure of SSAT in the external model.

The present invention provides following technical scheme according to above-mentioned technical conceive and result of study.

The present invention at first will assess in the potential SSAT path and mainly or only regulate the acetylizad specific drugs material standed for of N-through SSAT, and concrete grammar is following:

1) 37 ℃ of solubleness evaluations of hatching altogether of substrate, inhibition solution and tissue or cell;

2) substrate, inhibition solution are to the tissue of cryopreservation or the toxicity assessment of cell;

3) set up external model according to the method for CYP path phenotype in the U.S. FDA 2007 external drug metabolism guides and comprise application NAT1, NAT2 path specific inhibitor, confirm the specificity of NAT1, NAT2 selective depressant, see table 1.

Table 1, SSAT regulate the acetylizad specific drugs material standed for of N-

Be used to detect the N of Mammals spermine/spermine ¹The method of-acetyltransferase activity, it may further comprise the steps:

1) hatches a certain amount of a kind of medicine (that is: SSAT substrate) Mammals;

2) obtain certain tissue, cell or body fluid etc. from Mammals and analyze sample;

3) the acetylize metabolite in the check and analysis sample, there is be associated (positive correlation) in the acetylize metabolite with the SSAT activity.

The present invention realizes through following technical scheme:

1) described Mammals comprises people or other animals;

Described other animals comprise in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc. one or more;

In preferred people, mouse, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.;

Described hatching is meant through medicine (that is: SSAT substrate) and mammalian tissues, cell or body fluid and hatches altogether;

Described medicine (that is: SSAT substrate) is the L-DOPA, the dosage range of SSAT substrate: 1～10mg/kg, preferably 3～6mg/kg;

2) described analytic sample is to derive from mammiferous humoral sample that 0～24h hatches altogether or in the cell sample one or more, the mammalian cell that preferred 10～60min is hatched altogether, the further preferred rat hepatocytes of mammalian cell;

Described humoral sample is to comprise in blood, urine or the saliva one or more;

The preferred L-DOPA of the mammiferous humoral sample that described 0～24h is hatched altogether gets into and collects urine specimen in the mammalian body in 2～24h; Further preferably the L-DOPA gets into and collects urine specimen in the mammalian body in the 8h; Preferably the L-DOPA gets into and collects urine specimen in the mammalian body in the 4h again, most preferably is to collect urine specimen in the 2h in the L-DOPA entering mammalian body;

3) existence of acetylize metabolite is the active indication of Mammals SSAT in the analytic sample, and there is be associated with the SSAT activity (positive correlation) in the acetylize metabolite;

Acetylize metabolite method in the described check and analysis sample is the measured quantity according to acetylize metabolite in the Mammals sample; Making typical curve and confirm SSAT in the intravital activity of Mammals, can be to comprise in chromatographic detection method, radio-labeling, mass spectrum, infrared, near infrared or ultraviolet detection etc. one or more by multiple technologies;

Described chromatographic detection method is to comprise that gas-chromatography, high performance liquid chromatography (are called for short: HPLC), in thin-layer chromatography, specific antibody chromatogram or the antibody affinity chromatography etc. one or more;

Described positive correlation is meant that the measured quantity of acetylize metabolite is high more, and SSAT is high more in the intravital activity of Mammals.

Concrete experimentation of the present invention is following:

1, material and method:

Reagent: methyl alcohol, dimethyl sulfoxide (DMSO) (are called for short: DMSO), water, formic acid, hydrochloric acid, trypan blue solution, tetramethyl-azo azoles salt, Krebs-Henslaite damping fluid.

1.1 substrate: amantadine, spermidine, para-aminosalicylic acid, 4-benzaminic acid, sulfamethoxazole (are called for short: SMZ).

1.2 NAT1 and NAT2 pathway inhibitor: (±) methotrexate, paracetamol, Quercetin.

1.3 N-acetylize metabolite: N-ethanoyl-amantadine (AA), N-ethanoyl-sulfamerazine.

1.4 interior mark: N-ethanoyl-d ₃-amantadine, N-ethanoyl-sulfamerazine-d ₄

2, the former liver cell preparation of freezing female mouse

Adopt the rat liver cell, cell adopts the Celsis ex vivo technique to obtain stored under refrigeration in liquid nitrogen.Use the day before yesterday that the cryopreserved hepatocytes water-bath is thawed, process suspension-s with substratum, centrifugal, with the Krebs-Hensleite damping fluid (abbreviation: KHB) suspendible again, as the liver cell storing solution;

Before induction experiment, hepatocellular concentration is active with the active determination of trypan blue staining rat hepatocytes that adopts.The cell storing solution adds KHB adjustment cell concn to target level;

It is active before plating, to measure rat hepatocytes with the trypan blue method of exclusion earlier.

3, formulations prepared from solutions

Substrate solution preparation: para-aminosalicylic acid, para-amino benzoic acid, sulfamerazine, amantadine, spermidine, L-DOPA;

Inhibitor solution preparation: (±) methotrexate, paracetamol, Quercetin.

(1) solubleness evaluation

In substrate, the suppressor factor aqueous solution, add KHB, add or do not add DMSO, measure its final concentration.

Prepare each chemical storing solution, be diluted to test fluid with preheating KHB then.Test fluid is prepared into 3 concentration, and HPLC/UV measures its optical density, makes typical curve with optical density-concentration, obtains linear regression relation

(2) mtt assay rat hepatocytes cytotoxicity detects

The employing mtt assay is used to detect substrate and whether suppressor factor has cytotoxicity to refrigerating former mouse liver cell 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 simultaneously, altogether hatching.Follow preincubate, every hole adds the KHB that contains equivalent MTT, again hatching.After the hatching, remove substratum, add the DMSO dissolving, then use the deck vibrator hydrotropy, measure the optical density of solution with microplate reader 540nm.

(3) measure rat hepatocytes N-acetylize

Adopt 24 well culture plate vitro culture, each substrate or material standed for and NAT1 or NAT2 suppressor factor are hatched separately or hatched altogether, and be parallel in duplicate.

Add certain density suppressor factor or solvent control to culture plate.Each hole adds active liver cell storing solution then, hatches altogether.Follow preincubation, add the mixture of each substrate, material standed for or surrogate respectively, continue to hatch.Cultivate when finishing, add ice methyl alcohol stopped reaction in every hole.

Because solubility limit, L-DOPA is by independent cultivation, rather than mixes with other four material standed fors.Parallel two aliquot sample in every hole before LC/MS and LC/MS/MS analysis potential N-acetylize metabolite, are stored in the bottle and preserve.

(4) LC/MS and LC/MS/MS method are to the bioanalysis of N-acetylize metabolite

The N-acetylize metabolite of amantadine and sulphamethazine in this research employing 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.The QC sample concentration is respectively: N-acetyl amantadine 0.2,80 and 160ng/ml, N-acetylsulfanilamide diformazan pyrimidine 2800,1600ng/ml.

Owing to when this research is carried out, still do not have available metabolite reference standard, spermidine, para-aminosalicylic acid, para-amino benzoic acid, L-DOPA N-acetylize metabolite have been monitored with the method for target spot metabolite identification.Derive from vitro culture rat hepatocytes sample and at first be used to scan the medicine prototype, carry out the scanning of potential N-acetylize metabolite then, the segmental precursor of parent scan feature carries out directed multiple-reaction monitoring simultaneously.

4, result and analysis

(1) drug candidate, substrate and suppressor factor are water-soluble

Assessed water-soluble when the liver cell incubation buffer reaches the target final concentration of substrate in the KHB damping fluid, suppressor factor.When medicine is soluble, can add dimethyl sulfoxide (DMSO), methyl alcohol or 0.5MHCl hydrotropy, detect experimental concentration with linear UV absorption process and assess solubility promoter.Amantadine, spermidine are soluble.

(2) cytotoxicity of cryopreserved primary rat hepatocyte

Substrate add or do not add NAT1, the NAT1/NAT2 suppressor factor does not have cytotoxicity to the mouse liver cell, except amantadine at maximum concentration 2645 μ M (cytotoxicity is arranged).Three suppressor factor 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 the rat hepatocytes

NAT1 specific inhibitor Rheumatrex concentration 50 μ M, NAT2 specific inhibitor acetaminophen concentration 2144 μ M, the N-acetylize is about 0.15% when hatching finishes.

Inhibition NAT1 and the NAT2 coexistence 60min that is formed on of metabolite is suppressed 28%.When having NAT1 and NAT2 suppressor factor to exist, the 54%N-acetylize is suppressed.

(4) LC/MS/MS quantitative analysis N-acetyl-sulphamethazine and N-ethanoyl-amantadine

N-acetylsulfanilamide diformazan pyrimidine-D is adopted in N-acetyl-sulphamethazine and ethanoyl-amantadine quantitative analysis 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 LC/MS/MS quantitative analysis is proofreaied and correct matched curve and is the origin-excluded quadratic regression equation, and all concentration are used the 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 respectively Fig. 1, Fig. 2.

(5) the N-acetylize of rat hepatocytes SSAT positive control

Adopt two positive controls of SSAT substrate in the research: amantadine and spermidine.In rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor and share, or the common specific inhibitor of NAT1 and NAT2, independent respectively assessment N-acetylizing.

NAT1 specific inhibitor methotrexate concentration is 50 μ M.NAT2 specific inhibitor acetaminophen is owing to solubility limit, and concentration is 2144 μ M.The common suppressor factor Quercetin of NAT1 and NAT2 concentration is 480 μ M.N-acetylize metabolite derives from rat hepatocytes and amantadine (267 μ M and 2670 μ M) is hatched altogether in the rat hepatocytes sample, and N-acetyl metabolite amount is too low and fail to detect.Concentrated rat hepatocytes and 267 μ M amantadines are hatched sample and still can not be detected N-ethanoyl-amantadine.And the amantadine residuum does not almost change when hatching.The result is consistent with BRI No.BIM-2009-001 result of study.In rat urine, be checked through low-level N-acetylize-amantadine in the research in vivo.

Former medicine residuum ratio calculates through following formula:

Detected result shows: the N-acetylize metabolite of spermidine forms appearance fast at 0.Quantity was near 14% of the former medicine quantity that adds when hatching finished.Existence does not suppress this acetylization reaction with NAT2 suppressor factor (2144 μ M acetaminophen) because of NAT1 suppressor factor (50 μ M methotrexate).Yet 26% acetylization reaction is suppressed when having NAT1 suppressor factor (50 μ M methotrexate) merely, uses 480 μ M Quercetins also can produce restraining effect, and suppressor factor acts on NAT1 and two paths of NAT2.Former medicine is residual not to be changed.Meta-bolites, metabolism suppress, the remaining discordance of former medicine can solve through quantitative analysis.

SSAT substrate positive control and NAT1 and NAT2 suppressor factor test-results are illustrated in, and SSAT substrate spermidine N-acetylize can take place in the female rats liver cell model.The effect of N-ethanoyl is not united inhibition (methotrexate 50 μ M and acetaminophen 2144 μ M) by NAT1 suppressor factor and NAT2 suppressor factor.Yet it is influential to use methotrexate 50 μ M or acetaminophen 2144 μ M separately.Quercetin adopts concentration 480 μ M, in high concentration like this non-specific retarding effect possibly take place.Confirming further that in this research the Optimization Model parameter comprises inhibitor concentration, is one of following direction of this project.

(6) acetylizing of rat hepatocytes substrate L-DOPA

Adopt SSAT substrate material standed for L-DOPA in the research; In rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor and share; Or the common specific inhibitor of NAT1 and NAT2, independent respectively assessment N-acetylizing.

The result shows: the acetylize metabolite increases along with incubation time prolongs generation.When the L-DOPA that adds is 200 μ M, produce 0.5% metabolite; When the L-DOPA that adds is 20 μ M, produce 5.2% metabolite, there is the saturated or self-retarding effect of reaction in prompting.When the L-DOPA that adds was 20 μ M, former medicine residual quantity was lower.The common suppressor factor of NAT1 and NAT2 (Quercetin, 480 μ M) is arranged, and when existing, metabolite forms significantly and is suppressed (＞90%), and NAT1 suppressor factor (methotrexate, 50 μ M) and NAT2 suppressor factor (acetaminophen, 2144 μ M) exist, and metabolite forms and is not suppressed.

(7) conclusion

Show from above result, in female rats liver cell model, can produce NAT1 and NAT2 substrate N-acetylizing, use NAT1 suppressor factor methotrexate and the acting in conjunction of NAT2 suppressor factor acetaminophen and can suppress the N-acetylization reaction.On the contrary, there is the N-ethanoyl effect that can not change SSAT substrate spermidine simultaneously in these two suppressor factor, have also proved the specificity of these two NAT suppressor factor.

The result shows that also the L-DOPA is better than NAT1 and NAT2 the SSAT selectivity, and the undetected acetylizing of known SSAT substrate amantadine possibly be because sample is different.The SSAT rise is proportionate with cancer, so the L-DOPA can be used as the cancer markers that the SSAT rise is regulated.

(4) technology speciality

The present invention provides a kind of new detection method for prevention, diagnosis, detection, protection, treatment and research tumour; Promptly, found a kind of detection Mammals spermidine/spermine N through mainly or only regulating the acetylizad specific substrate of N-in the assessment potential SSAT path through SSAT ¹The method of-acetyltransferase activity, thus carried out improvement, improved at aspects such as the discovery morning of tumour, diagnosis morning and early treatments, to overcome the above-mentioned defective that prior art exists.

That is to say; The present invention is through research and theory studies such as experimentation on animals, clinical trials; Provide a kind of and new spermidine/spermine substrate acetylated form has been detected the active quantitative technique of SSAT; Acetylize metabolite through detecting the SSAT substrate is analyzed the active method of SSAT; Wherein: the SSAT substrate is a levodopa, and the part metabolism of levodopa is can produce acetylize metabolite N-ethanoyl-L-DOPA through inducing of SSAT.The L-DOPA is better than NAT1 and NAT2 the SSAT selectivity, and the SSAT rise is proportionate with cancer, so the L-DOPA can be used as the cancer markers that the SSAT rise is regulated.

The inventive method is safe and effective, and practicality is stronger, and its working method is convenient and swift, and is easy to use, and positive rate is high, and effect is remarkable, can be used for preventing, diagnose, detect, protect, treating and study the supplementary means of all kinds of tumours.

The present invention studies cancer markers targetedly; Found a kind of new detection method; Made beyond thought achievement; It is safe in utilization; The detection effect of preventing, diagnose, detect, protect, treat and study all kinds of tumours is obvious; And use range is wide especially, therefore applies easily, 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 provides new detection means for research and development are antitumor; Having important value to improving and improving existing medical level, is the detection method that is used to prevent, diagnose, detect, protect, treat and study aspects such as antitumor and directly related disease.

Description of drawings

Fig. 1 is N-ethanoyl in the cryopreserved primary rat hepatocyte-sulphamethazine LC/MS/MS quantitative correction typical curve;

Wherein, * expression standard substance, ◇ representes the gas phase sample;

Fig. 2 is N-ethanoyl in the cryopreserved primary rat hepatocyte-amantadine LC/MS/MS quantitative correction typical curve;

Wherein, * expression standard substance, ◇ representes the gas phase sample;

Fig. 3 is that amantadine concentration 267 μ M (reaching the standard grade), 2670 μ M (rolling off the production line) and rat hepatocytes are in the external former medicine residual content in back of hatching altogether;

Wherein, * expression standard substance, ◇ representes the gas phase sample;

Amantadine: amantadine; Amethopterin: methotrexate;

Acetaminophen: paracetamol; Quercetin: Mongolian oak skin (Huang) element;

Remaining: residual; Time: time;

Min: minute.

Fig. 4 is that 550 μ M spermidines and rat hepatocytes are at the external back N-acetylize spermidine of hatching altogether;

Spermidine; Spermidine, spermidine; Amethopterin: methotrexate;

Acetaminophen: paracetamol; Quercetin: Mongolian oak skin (Huang) element;

As parent at T=0 accounts for the per-cent of female medicine when zero; The time time;

Min: minute.

Fig. 5 is that 550 μ M spermidines and rat hepatocytes are in the external former medicine residual content in back of hatching altogether;

Spermidine: spermidine, spermidine; Amethopterin: methotrexate;

Acetaminophen: paracetamol; Quercetin: Mongolian oak skin (Huang) element;

Remaining: residual; Time: time;

Min: minute.

Fig. 6 is the external back N-acetylize L-DOPA formation amount of hatching altogether of 200 μ ML-DOPA and rat hepatocytes;

L-Dopa: levodopa; The amethopterin methotrexate;

Quercetin: Mongolian oak skin (Huang) element; Time: time;

As parent at T=0: the per-cent that when zero, accounts for female medicine; Min: minute.

Fig. 7 is the external former medicine residual quantity in back of hatching altogether of 200 μ ML-DOPA and rat hepatocytes;

L-Dopa: levodopa; Amethopterin: methotrexate;

Quercetin: Mongolian oak skin (Huang) element; Remaining: residual;

Time: time; Min: minute.

Fig. 8 is the external N-acetylize L-DOPA formation amount of hatching altogether of 20 μ ML-DOPA and rat hepatocytes;

L-Dopa: levodopa; The amethopterin methotrexate;

Quercetin: Mongolian oak skin (Huang) element; Time: time;

As parent at T=0 accounts for the per-cent of female medicine when zero; Min: minute.

Figure 92 0 μ ML-DOPA and rat hepatocytes be external hatches former medicine residual quantity altogether;

L-Dopa: levodopa; The amethopterin methotrexate;

Quercetin: Mongolian oak skin (Huang) element; Remaining: residual;

Time: time; Min: minute.

Embodiment

The present invention has studied a kind of medical detection technique, promptly a kind of spermidine/spermine N ¹-Transacetylase (be called for short: SSAT) active method, provide a kind of and analyzed the active method of SSAT through the acetylize metabolite that detects the SSAT substrate, be convenient to the convenient of medical industry and safe handling.

Below further set forth and illustrate with regard to the concrete experimentation of implementing.

Concrete experimentation of the present invention is following:

1, material and method:

Reagent

Methyl alcohol (EMD company, HPLC level); Dimethyl sulfoxide (DMSO) (be 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 all concentration of substrate of this research and revise according to dated chemical purity.

1.2 NAT1 and NAT2 pathway inhibitor

Table 3, NAT1 and NAT2 pathway inhibitor

Remarks: be applied to all concentration of substrate of this research and revise according to dated chemical purity

1.3 N-acetylize metabolite: following metabolite is as reference standard correction in this research LC/MS/MS bioanalysis

N-acetylize metabolite in table 4, the LC/MS/MS bioanalysis

1.4 interior mark

Mark in table 5, the LC/MS/MS bioanalysis

Remarks: purity does not have correction calculation.

2, the former liver cell preparation of freezing female mouse

(the Wistar rat, female, 200～300g) liver cells, cell adopt the Celsis ex vivo technique to obtain stored under refrigeration in liquid nitrogen to adopt 20 rats.Use the day before yesterday that cryopreserved hepatocytes is thawed 37 ℃ of water-baths, process suspension-s with substratum, centrifugal 10 minutes of 100xg, with the Krebs-Hensleite damping fluid (abbreviation: KHB) suspendible again, as the liver cell storing solution.

Before induction experiment, hepatocellular concentration and the active determination of trypan blue staining that adopts, the rat hepatocytes activity is 95% (Hu4161, Hu4198 and Hu4201) in preliminary experiment.Be confirmed as 93% at the employed mouse hepatocyte activity of repeated experiments (Hu4227).Through measuring hepatocyte activity, the cell storing solution adds KHB adjustment cell concn to target level 0.51x10 ³/ ml is equivalent to 2.5X10 ⁵Viable cell/0.49ml.

Table 6, substrate, inhibitor solution preparation

3, formulations prepared from solutions

(1) solubleness evaluation

37 ℃ add the Krebs-Henslaite damping fluids in substrate, the suppressor factor aqueous solution, add or do not add 0.1～0.2%DMSO, measure its final concentration.

Prepare each chemical storing solution, use preheating KHB (37 ℃) to be diluted to test fluid then.Test fluid is prepared into 3 concentration, and HPLC/UV measures its optical density, makes typical curve with optical density-concentration, obtains linear regression relation

(2) mtt assay rat hepatocytes cytotoxicity detects

Adopt 96 orifice plates, liver cell (0.5X106/ml) respectively with substrate, the material standed for (0,1/100,1/50,1/20,1/10,1/5,1/2,1 times higher target level) of different concns, add NAT1 or NAT2 or NAT1 and NAT2 simultaneously, hatch 15m altogether.Then preincubate is 10 minutes, and every hole adds the KHB (final concentration 0.5mg/ml) that contains equivalent MTT, hatches 30m again.After the hatching, remove substratum, add 100 μ l DMSO dissolving, then use the deck vibrator hydrotropy, measure the optical density of solution with microplate reader 540nm.

(3) measure rat hepatocytes N-acetylize

Adopt 24 well culture plate vitro culture, 37 ℃ of incubator temperature, contain 5%CO ₂, 95% air.Each substrate or material standed for and NAT1 or NAT2 suppressor factor are hatched separately or hatched altogether, and be parallel in duplicate.

Add certain density suppressor factor or solvent control (5 μ L) to culture plate.Each hole adds and contains about 2.5X10 then ⁵Active liver cell storing solution 0.49ml is hatched 10min altogether.Then preincubation 10min adds the mixture of 5 each substrate of μ L, material standed for or surrogate respectively, continues to hatch 0,30 and 60min.Cultivate when finishing, add 0.5ml ice methyl alcohol stopped reaction in every hole.

Because solubility limit, L-DOPA is by independent cultivation, rather than mixes with other four material standed fors.Parallel two aliquot sample in every hole before LC/MS and LC/MS/MS analysis potential N-acetylize metabolite, are stored in the 1.5ml bottle, are kept at-80 ℃ (72 ℃～-88 ℃).

(4) LC/MS and LC/MS/MS method are to the bioanalysis of N-acetylize metabolite

The N-acetylize metabolite of amantadine and sulphamethazine in this research employing 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.The QC sample concentration is respectively: N-acetyl amantadine 0.2,80 and 160ng/ml; N-acetylsulfanilamide diformazan pyrimidine 2800,1600ng/ml.

Owing to when this research is carried out, still do not have available metabolite reference standard, spermidine, para-aminosalicylic acid, para-amino benzoic acid, L-DOPA N-acetylize metabolite have been monitored with the method for target spot metabolite identification.Derive from vitro culture rat hepatocytes sample and at first be used to scan the medicine prototype, carry out the scanning of potential N-acetylize metabolite then, the segmental precursor of parent scan feature carries out directed multiple-reaction monitoring simultaneously

4, result and analysis

(1) drug candidate, substrate and suppressor factor are water-soluble

Water-soluble when the liver cell incubation buffer reaches the target final concentration of substrate when having assessed 37 ℃ in the KHB damping fluid, suppressor factor.When medicine is soluble, can in 0.1%～0.2%KHB, add dimethyl sulfoxide (DMSO), methyl alcohol or 0.5MHCl hydrotropy, detect experimental concentration with linear UV absorption process and assess solubility promoter.Amantadine, spermidine are soluble.

(2) cytotoxicity of cryopreserved primary rat hepatocyte

Inhibition NAT1 and the NAT2 coexistence 60min that is formed on of metabolite is suppressed 28% (Fig. 3 and table 7).When having NAT1 and NAT2 suppressor factor to exist, the 54%N-acetylize is suppressed.

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

(5) the N-acetylize of rat hepatocytes SSAT positive control

NAT1 specific inhibitor methotrexate concentration is 50 μ M.NAT2 specific inhibitor acetaminophen is owing to solubility limit, and concentration is 2144 μ M.The common suppressor factor Quercetin of NAT1 and NAT2 concentration is 480 μ M.N-acetylize metabolite derives from rat hepatocytes and amantadine (267 μ M and 2670 μ M) is hatched altogether in the rat hepatocytes sample, and N-acetyl metabolite amount is too low and fail to detect.Concentrated rat hepatocytes and 267 μ M amantadines are hatched sample and still can not be detected N-ethanoyl-amantadine.And the amantadine residuum does not almost change (Fig. 3 and table 7) when hatching.The result is consistent with BRI No.BIM-2009-001 result of study.In rat urine, be checked through low-level N-acetylize-amantadine in the research in vivo.

Former medicine residuum ratio calculates through following formula:

Acetylized admantadine and former medicine residual quantity that table 7, different initial substrate concentration form

Annotate: lower limit of quantitation=0.1ng/mL

Detected result shows: the N-acetylize metabolite of spermidine forms appearance fast at 0.Quantity was near 14% of the former medicine quantity that adds when hatching finished.Existence does not suppress this acetylization reaction with NAT2 suppressor factor (2144 μ M acetaminophen) because of NAT1 suppressor factor (50 μ M methotrexate).Yet 26% acetylization reaction is suppressed when having NAT1 suppressor factor (50 μ M methotrexate) merely, uses 480 μ M Quercetins also can produce restraining effect, and suppressor factor acts on NAT1 and two paths of NAT2 (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 solve through quantitative analysis.

Table 8, rat hepatocytes and SSAT substrate 550 μ M spermidines

Add or do not add NAT1 and the NAT2 suppressor factor is external hatch altogether after, N-acetylize metabolite and former medicine residual content

(6) acetylizing of rat hepatocytes substrate L-DOPA

Adopt SSAT substrate material standed for L-DOPA (20 μ M and 200 μ M) in the research; In rat hepatocytes, add or do not add independent NAT1 specific inhibitor or NAT1 specific inhibitor and NAT2 specific inhibitor and share; Or the common specific inhibitor of NAT1 and NAT2, independent respectively assessment N-acetylizing.

The result shows: the acetylize metabolite increases along with incubation time prolongs generation.When the L-DOPA that adds is 200 μ M, produce 0.5% metabolite; When the L-DOPA that adds is 20 μ M, produce 5.2% metabolite, there is the saturated or self-retarding effect of reaction in prompting.When the L-DOPA that adds was 20 μ M, former medicine residual quantity was lower.The common suppressor factor of NAT1 and NAT2 (Quercetin, 480 μ M) is arranged when existing, metabolite forms significantly and is suppressed (＞90%); And NAT1 suppressor factor (methotrexate, 50 μ M) and NAT2 suppressor factor (acetaminophen, 2144 μ M) exist; Metabolite forms and is not suppressed (Fig. 6,7,8,9, table 9).

Table 9, L-DOPA add or do not add NAT1 and the NAT2 suppressor factor is external hatch altogether after, N-acetylize metabolite and former medicine residual content

Remarks 1: suppose metabolite and former medicine plasma efficient

(7) conclusion

In the present invention, the embodiment of above-described embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting scope of the present invention.Through embodiment the present invention is described in detail below.

Embodiment 1,

(the Wistar rat, female, 200-300g) liver cell, cell adopt the Celsis ex vivo technique to obtain stored under refrigeration in liquid nitrogen to adopt 20 rats.Use the day before yesterday that cryopreserved hepatocytes is thawed 37 ℃ of water-baths, process suspension-s with substratum, centrifugal 10 minutes of 100xg is with Krebs-Hensleite damping fluid (KHB) suspendible again.Adopt 24 orifice plate vitro culture, 37 ℃ of culture temperature, contain 5%CO ₂, 95% air, each hole adds and contains about 2.5 * 10 ⁵Active liver cell storing solution 0.49ml is hatched 10min altogether.Follow preincubation 10min.One group of adding L-DOPA is 200 μ M, suppressor factor Quercetin 480 μ M in the liver cell solution; One group adds the L-DOPA is 200 μ M, suppressor factor methotrexate 50 μ M; One group adds the L-DOPA is 200 μ M, suppressor factor acetaminophen 2144 μ M; The mixing solutions 5 μ l of each substrate, inhibition are to culture plate; Continue to hatch 0,30 and 60min.Cultivate when finishing, add 0.5ml ice methyl alcohol stopped reaction in every hole.Every group duplicate.Measure the result and see table 10.

Table 10,200 μ M L-DOPA are adding or are not adding external back N-acetylize metabolite and the former medicine residual content of hatching altogether of NAT1 and NAT2 suppressor factor

Embodiment 2,

(the Wistar rat, female, 200～300g) liver cells, cell adopt the Celsis ex vivo technique to obtain stored under refrigeration in liquid nitrogen to adopt 20 rats.Use the day before yesterday that cryopreserved hepatocytes is thawed 37 ℃ of water-baths, process suspension-s with substratum, centrifugal 10 minutes of 100xg is with Krebs-Hensleite damping fluid (KHB) suspendible again.Adopt 24 orifice plate vitro culture, 37 ℃ of culture temperature, contain 5%CO ₂, 95% air, each hole adds and contains about 2.5 * 10 ⁵Active liver cell storing solution 0.49ml is hatched 10min altogether.Follow preincubation 10min.One group of adding L-DOPA is 20 μ M, suppressor factor Quercetin 480 μ M in the liver cell solution; One group adds the L-DOPA is 200 μ M, suppressor factor methotrexate 50 μ M; One group adds the L-DOPA is 200 μ M, suppressor factor acetaminophen 2144 μ M; The mixing solutions 5 μ l of each substrate, inhibition are to culture plate; Continue to hatch 0,30 and 60min.Cultivate when finishing, add 0.5ml ice methyl alcohol stopped reaction in every hole.Every group duplicate.Measure the result and see table 11.

Table 11,20 μ M L-DOPA are adding or are not adding external back N-acetylize metabolite and the former medicine residual content of hatching altogether of NAT1 and NAT2 suppressor factor

Claims

1. one kind is used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described method may further comprise the steps:

1) hatches a certain amount of a kind of medicine Mammals;

3) the acetylize metabolite in the check and analysis sample, there are positive correlation in acetylize metabolite and SSAT activity.

2. according to claim 1ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described Mammals comprises people or other animals.

3. according to claim 2ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described other animals comprise one or more in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey.

4. according to claim 3ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described other animals are one or more in people, mouse, rat, monkey, pig, rabbit or the dog.

5. according to claim 4ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described other animals are one or more in people, rat or the monkey.

6. according to claim 1ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described medicine is the SSAT substrate, is the L-DOPA.

7. according to claim 1ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described hatching is meant through medicine and mammalian tissues or cell and hatches altogether.

8. describedly be used to detect spermidine/spermine N according to claim 6 or 7 ¹The method of-acetyltransferase activity is characterized in that, the dosage range of described medicine is 1～10mg/kg.

9. according to claim 8ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the dosage range of described medicine is 3～6mg/kg.

10. according to claim 1ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described analytic sample is to derive from mammiferous humoral sample that 0～24h hatches altogether or in the cell sample one or more.

11. according to claim 10ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described analytic sample is to derive from mammiferous humoral sample that 10～60min hatches altogether or in the cell sample one or more.

12. according to claim 11ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described analytic sample is to derive from the rat hepatocytes that 10～60min is hatched altogether.

13. according to claim 10ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 2～24h in the mammalian body.

14. according to claim 14ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 8h in the mammalian body.

15. according to claim 15ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 4h in the mammalian body.

16. according to claim 16ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 2h in the mammalian body.

17. describedly be used to detect spermidine/spermine N according to claim 1 or 10 ¹The method of-acetyltransferase activity is characterized in that, described humoral sample is to comprise in blood, urine or the saliva one or more.

18. according to claim 18ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 2～24h in the mammalian body.

19. according to claim 19ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 8h in the mammalian body.

20. according to claim 20ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 4h in the mammalian body.

21. according to claim 21ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the mammiferous humoral sample that described 0～24h is hatched altogether is that the L-DOPA gets into the interior urine specimen of collecting of 2h in the mammalian body.

22. according to claim 1ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the acetylize metabolite method in the described check and analysis sample is the measured quantity according to acetylize metabolite in the Mammals sample, makes typical curve and confirms that SSAT is in the intravital activity of Mammals.

23. according to claim 23ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, the determination techniques of acetylize metabolite content is to comprise in chromatographic detection method, radio-labeling, mass spectrum, infrared, near infrared or the ultraviolet detection one or more in the described Mammals sample.

24. according to claim 24ly be used to detect spermidine/spermine N ¹The method of-acetyltransferase activity is characterized in that, described chromatographic detection method is to comprise in gas-chromatography, high performance liquid chromatography, thin-layer chromatography, specific antibody chromatogram or the antibody affinity chromatography one or more.