CN101380323A - Pharmaceutical composition and use thereof - Google Patents
Pharmaceutical composition and use thereof Download PDFInfo
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- CN101380323A CN101380323A CNA2008101968758A CN200810196875A CN101380323A CN 101380323 A CN101380323 A CN 101380323A CN A2008101968758 A CNA2008101968758 A CN A2008101968758A CN 200810196875 A CN200810196875 A CN 200810196875A CN 101380323 A CN101380323 A CN 101380323A
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Abstract
The invention relates to a pharmaceutical composite which contains isoniazide, tiopronin or sodium salt thereof that are taken as the active constituents, and a pharmaceutical carrier and/or an excipient, wherein, the mass ratio of the isoniazide and the tiopronin is 1-10:1. The tiopronin has specific action on inhibiting hepatic CYP2E1, and can specifically treat liver damage caused by the isoniazide which is an antituberculotic, and purposefully reduces the toxic and side effect of the isoniazide on the liver. The pharmaceutical composite not only further expands the pharmaceutical value of the tiopronin which is an inhibitor of the hepatic CYP2E1, but also contributes to the safe clinical application of the related pharmaceutical (isoniazide). Therefore, the pharmaceutical composite has important medical value and social and economic benefits.
Description
One, technical field
The present invention relates to a kind of pharmaceutical composition and application thereof, described pharmaceutical composition contains as the isoniazid of active component and tiopronin or its sodium salt and pharmaceutically suitable carrier and/or excipient; Can be used for tuberculotherapy.
Two, background technology
Tiopronin (English name Tiopronin, chemical name: mercapto-propionyl-glycin), its molecular formula: C
5H
9NO
3SH, molecular weight: 163.2, structural formula is as follows:
Liver is that body carries out all kinds of materials and transforms metabolic major organs, expresses the metabolic enzyme system of multiple participation material, wherein the most important thing is Cytochrome P450 (being called for short CYPs).CYPs is a superfamily enzyme system of containing multiple hypotype, and multiple endogenous material of metabolism (the synthetic material of body self metabolism) and exogenous material (comprising medicine, environmental poisonous substance etc.) mainly are expressed in liver.In CYPs, hypotype 2E1 (CYP2E1) main metabolic micromolecular compound comprises multiple medicine, halogenated hydrocarbon, alcohols, ketone and endogenous material such as arachidonic acid, fatty acid etc.
CYP2E1 is significant aspect toxicology, and especially aspect hepar damnification, its catalysis metabolic drug causes the concrete mechanism of action of liver function damage mainly to comprise following three aspects.The first, CYP2E1 participates in drug metabolism directly, and the oxidation medicine generates toxic metabolite, as the ntipyretic analgesic medicine acetaminophen (acetaminophen, APAP).The trade name of acetaminophen includes bufferin, panadol, acetaminophen, acetaminophen, Tylenol Regular Strength, acetaminophen, acetaminophen, panadol, tylenol etc.After APAP enters in the body, can by the CYP2E1 metabolism generate toxicity intermediate product acetophenone quinone imines (N-acetyl-p-benzoquinone imine, NAPQI).NAPQI can combine with macromolecular substances such as nucleic acid, protein, then cause cell injury [Lee, S.S., J.T.Buters, T.Pineau, P.Fernandez-Salguero, F.J.Gonzalez, Role of CYP2El in the hepatotoxicity ofacetaminophen.J Biol Chem, 1996.271 (20): p.12063-7.].Second, CYP2E1 can combine with liver albumen because of the intermediate product that the oxidation medicine is produced and form new antigen, thereby the immunoreation of bringing out body causes the liver major injury, as anesthetis halothane [Wang Chunhua, establish an army, Lang Zhenwei, Wu Yu, Yang Yanjie, record the winter, Dang Xiaoyan, the progress of cytochrome C YP II El and hepatic disease relation. world Chinese digests magazine, and 2004.12 (4): p.950-4.].The 3rd, because of CYP2E1 has stronger NADPH (NADPH) oxidase active, but event CYP2E1 is catalysis medicine or its metabolite generation activating oxide (reactive oxygen species also, ROS) or free radical [Jaeschke, H., G.J.Gores, A.I.Cederbaum, J.A.Hinson, D.Pessayre, J.J.Lemasters, Mechanisms of hepatotoxicity.Toxicol Sci, 2002.65 (2): p.166-76.], as antituberculotics isoniazid or its metabolite hydrazine.ROS or free radical can bring out a series of pathological processes and cause cell injury, as the unsaturated fatty acid of attacking in the membrane lipid causes that lipid peroxidation causes membrane structure impaired, energy metabolism impairment, but also Profilin matter is synthetic, and form the DNA addition product, finally cause hepatocyte death.
CYP2E1 is considered to influence the important determiner of body to various poisonous substance infringements and carcinogenecity sensitivity, especially the mediation hepar damnification aspect [Gonzalez, F.J., Role of cytochromes P450 inchemical toxicity and oxidative stress:studies with CYP2El.Mutat Res, 2005.569 (1-2): p.101-10.].Known, at the liver lobule, be positioned at that hepatocyte CYP2E1 expression exceeds 30 times than portal area CYP2E1 level around the central vein.Have hepatotoxic material or carcinogen and mainly show lobule central area (be central vein around) through the hepatic necrosis that CYP2E1 mediation produces.The high CYP2E1 expression of this explanation hepatocyte then is prone to the cell injury necrosis, be CYP2E1 expression height and the caused toxic damages degree of xenobiotics [Caro in close relations, A.A., A.I.Cederbaum, Oxidativestress, toxicology, and pharmacology of CYP2El.Annu Rev Pharmacol Toxicol, 2004.44:p.27-42.].Clinical trial also confirms, CYP2E1 expression of enzymes level in the drug-induced liver disease morbidity, play an important role [Guo Yanmei, Wang Qin, Cytochrome P450 2E1 enzyme and drug-induced liver disease. health professional education, 2006.24 (19): p.130-1.].
Isoniazid is applicable to a clinical line medicine of tuberculotherapy.Isoniazid was invented in nineteen fifty-two, and the invention of isoniazid makes treatment tuberculosis play the variation of essence.The sterilization characteristic of isoniazid is: it can suppress the synthetic of tulase bacterium wall mycolic acids composition, thereby make tubercule bacillus lose (the acidproof dyeing of multiple ability, proliferative ability, hydrophobicity) and dead, isoniazid can also combine with tulase thalline coenzyme, play the synthetic effect of interference DNA (deoxyribonucleic acid) and ribonucleic acid, thereby reached the purpose of killing tulase.And it is stronger to the strong tulase effect of metabolic activity.Isoniazid is as antituberculotics, and common service time is longer, and liver function injury is one of its main side effect.When patient's abnormal liver function, must drug withdrawal carry out liver protecting therapy.Result of study before us shows, isoniazid generates free radical through CYP2E1 catalysis metabolism and causes hepar damnification [YueJiang, Peng Ren-Xiu, Yang Jing, et al.CYP2E1 mediated isoniazid-inducedhepatotoxicity in rats.Acta Pharmacol Sin 2004; 25 (5): 699-704.].
People CYP2E1 gene contain altogether 11,413 base pairs (base pair, bp), 2788 bp in upstream, 599 bp in downstream contain 9 exons and typical TATA box.Rat CYP2E1 gene comprises 10,373 bp in coding region, 1530 bp in upstream, and 825 bp in downstream contain 9 exons and 8 introns.The CYP2E1 gene coding region has good conservative aspect kind, for the base sequence of upstream, transcription initiation position important area (140 bp), rat CYP2E1 gene and people are in full accord.Through immunosuppressant, immune quantitative and structural research confirm that all rat is the splendid animal of research CYP2E1 gene expression and function, but and with human CYP2E1 catalysis same reaction, be the good animal model [Morel that is used to study, G., B.Cossec, A.M.Lambert, S.Binet, Evaluation of rat hepatic 2E1 activity in function ofage, sex and inducers:choice of an experimental model capable of testing thehepatotoxicity of low molecular weight compounds.Toxicol Lett, 1999.106 (2-3): p.171-80.].
Three, summary of the invention
Problem to be solved by this invention provides a kind of pharmaceutical composition and application thereof, and described pharmaceutical composition can be used for tuberculotherapy, to reduce the side effect of drug induced injury liver.
The technical scheme that the present invention addresses the above problem is: a kind of pharmaceutical composition, contain isoniazid, tiopronin or its sodium salt and pharmaceutically suitable carrier and/or excipient as active component; Wherein the mass ratio of isoniazid and tiopronin is 1～10:1; Preferred 5:1.
The content of isoniazid is 50～300mg in the aforementioned pharmaceutical compositions.
Above-mentioned pharmaceutically suitable carrier and/or excipient are selected from carboxymethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, maltose, sucrose, medical starch, magnesium stearate, water for injection; Antioxidant (sodium sulfite, sodium sulfite, sodium pyrosulfite, ascorbic acid, cysteine hydrochloride, glycine, propylene glycol, thiourea); And in the osmotic pressure regulator (sodium chloride, glucose, mannitol, xylitol, potassium chloride) one or more.
The content of isoniazid is 0.01～10% in the aforementioned pharmaceutical compositions, and the content of tiopronin is 0.01～2%, and the content of pharmaceutical carrier and/or excipient is 88～99%, and above percentage ratio is mass ratio.
Pharmaceutical composition of the present invention can be injection such as sterile powder injection, lyophilized injectable powder or aqueous injection; Also can be oral formulations such as tablet, capsule, oral liquid, sustained-release preparation or granule.The preparation specification comprises 0.05g isoniazid, 0.1g isoniazid, 0.2g isoniazid, 0.3g isoniazid.
The application of pharmaceutical composition of the present invention in preparation treatment tuberculosis.
The usage and dosage of suggestion of the present invention:
Oral with capsule is example, and every capsules contains isoniazid 100mg, tiopronin 20mg (mass ratio of isoniazid and tiopronin can be 1～10:1, and the recommendation ratio is 5:1), and adult's suggestion consumption is 3 decoction being taken at a draught every day, 3～6 months courses of treatment, or follow the doctor's advice.With the lyophilized injectable powder injection is example, every contains isoniazid 100mg, (mass ratio of isoniazid and tiopronin can be 1～10:1 to tiopronin 20mg, the recommendation ratio is 5:1), adult's suggestion consumption is 1～2 of intramuscular injection once a day, or dilute posterior vein with glucose or normal saline and instil 2,1～2 month course of treatment, or follow the doctor's advice.
Isoniazid is as antituberculotics, and common service time is longer, and liver function injury is one of its main side effect.When isoniazid causes hepatic injury, present therapeutic scheme clinically, normally drug withdrawal and in addition general nonspecific drug treatment.In view of liver CYP2E1 is the key factor that isoniazid causes drug induced hepatic injury, the present invention is prepared into compositions with specific inhibitor tiopronin and the isoniazid of CYP2E1, can be effectively and specificity reduces isoniazid on the mechanism liver toxicity, be used for treatment lungy; Thereby the hepar damnification that occurs in the prophylactic process is not incured loss through delay original treatment of diseases.
Four, description of drawings
Fig. 1 illustrates that AH (Cytochrome P450 2E1 catalytic reaction) meets the Michaelis-Menten equation in concentration of substrate 4～800 μ mol/L scopes;
Fig. 2 A explanation tiopronin is concentration dependent and suppresses the CYP2E1 activity;
Fig. 2 B is the semilog mapping of Fig. 2 A, is used to calculate tiopronin IC
50Value (half-inhibition concentration);
Fig. 3 illustrates that tiopronin is the inducing action of concentration dependent antagonism isoniazid to CYP2E1;
Fig. 4 is a hepatic pathology slice map of the present invention, illustrates that tiopronin and isoniazid compositions group liver toxicity are starkly lower than the isoniazid list with organizing; A wherein: matched group (* 200); B and C: isoniazid group (* 200, * 400); D: isoniazid and tiopronin compositions group 1 (* 200). (hematoxylin-eosin staining);
Fig. 5 A and Fig. 5 B are the product molecule [M+H] of 8-hydroxyl deoxyguanosine (8-OH-dG)
+(m/z 284.3) and [M+Na]
+The mass spectrum of (m/z 306.1);
Fig. 6 is 8-hydroxyl deoxyguanosine (150mg/L) and the mass spectrum of deoxyguanosine (50mg/L) standard substance under the MRM analytical model.
Five, the specific embodiment
Embodiment 1: pharmaceutical composition of the present invention is made 1000 capsules by isoniazid 100 grams, tiopronin 20 grams, carboxymethyl cellulose 30 grams, sucrose 20 grams, medical starch 50 grams (or selecting other pharmaceutically suitable carrier and/or excipient such as maltose etc.).Its preparation method is: place centrifugal coating pelletizing machine material pot to form granule isoniazid, tiopronin and pharmaceutically suitable carrier and/or excipient mixture, and fill enteric coated capsule then, every capsules contains isoniazid 100mg, tiopronin 20mg.
Embodiment 2: pharmaceutical composition of the present invention is made 1000 capsules by isoniazid 300 grams, tiopronin 30 grams, ethyl cellulose 25 grams, sodium sulfite 0.15 gram, medical starch 50 grams.Its preparation method such as embodiment 1, every capsules contains isoniazid 300mg, tiopronin 20mg.
Embodiment 3: pharmaceutical composition of the present invention with maltose 50 grams (or selecting other pharmaceutical carriers and/or excipient such as sucrose, fructose etc.), adds the injection water to 1000ml by isoniazid 100 grams, tiopronin 50 grams, is distributed into 1000 and makes.Its preparation method is: tiopronin 100 grams and maltose 50 grams are dissolved in 1 liter of water for injection, and fully stirring and dissolving adds 0.01～0.1% injection activated carbon adsorption pyrogen, remove by filter active carbon, malleation microporous filter degerming, packing, every contains isoniazid 100mg, tiopronin 50mg.
Embodiment 4: pharmaceutical composition of the present invention with sucrose 100 grams, adds the injection water to 1000ml by isoniazid 100 grams, tiopronin 20 grams, is distributed into 1000 and makes.Its preparation method such as embodiment 3, every contains isoniazid 100mg, tiopronin 20mg.
It is animal model that the present invention uses rat, has observed the inside and outside rejection characteristic of tiopronin to CYP2E1, and has compared the compositions of isoniazid and tiopronin and isoniazid list with causing the hepar damnification situation.
1 tiopronin suppresses the experimentation of CYP2E1 activity influence
1.1 tiopronin suppresses the mensuration of constant to the CYP2E1 enzymatic activity
CYP2E1 mediation aniline hydroxylation reaction so AH is the probe enzyme of CYP2E1, is measured the AH activity and be can be used for indicating the CYP2E1 enzymatic activity.The AH activity increases with concentration of substrate, is the saturation kinetics feature, meets the Michaelis-Menten equation.K as calculated
m95% credibility interval of value is 24.91 to 41.61 μ mol/L, V
MaxValue is 0.39 μ mol/min/mg (Fig. 1).
According to experimental result, selecting aniline concentration is 40 μ mol/L, carries out tiopronin IC
50Value (half-inhibition concentration) determination experiment.The result as seen, tiopronin is concentration dependent and suppresses the AH activity, the semilog linear regression analysis shows tiopronin IC
50The value for 0.12mmol/L (Fig. 2 A, B).
1.2 tiopronin is induced the experiment in vitro research of CYP2E1 activity influence to isoniazid
The continuous lumbar injection isoniazid of rat (100mg/kg) 10 days, hepatomicrosome CYP2E1 activity is active (1.07 ± 0.21) the μ mol/min/g of being of AH, raises 3.8 times than blank group AH activity (0.28 ± 0.06) μ mol/min/g.Add the tiopronin of variable concentrations, isoniazid group CYP2E1 activity obviously is suppressed, and is concentration dependent (Fig. 3).
The comparative study of hepatotoxic effect of 2 tiopronins and isoniazid compositions liver toxicity and isoniazid list
Male Wistar rat, the SPF level, body weight 230～270g is available from Hubei Province's medical scientific institute zoopery center.Animal is divided into matched group, isoniazid group, isoniazid and tiopronin compositions group 1 (mass ratio of isoniazid and tiopronin is 5:1), isoniazid and tiopronin compositions group 2 (mass ratio of isoniazid and tiopronin is 10:1), isoniazid and tiopronin compositions group 3 (mass ratio of isoniazid and tiopronin is 2.5:1) at random.The continuous lumbar injection isoniazid of isoniazid group rat (100mg/kg) 21 days.Isoniazid and tiopronin compositions group 1, group 2, group 3, rats by intraperitoneal injection pharmaceutical composition (group 1: isoniazid 100mg/kg, tiopronin 20mg/kg; Group 2: isoniazid 100mg/kg, tiopronin 10mg/kg; Group 3: isoniazid 100mg/kg, tiopronin 40mg/kg) 21 days, capacity normal saline such as matched group injection.
Rat is injected to put to death in back 1 hour in last and gets blood, measures serum glutaminic acid aminotransferase (sALT), aspartate aminotransferase (sAST) and AH (CYP2E1) activity.Serum glutaminic acid aminotransferase (sALT), aspartate aminotransferase (sAST) assay method reference reagent box description.
With the matched group ratio, isoniazid rat blood serum glutamic acid aminotransferase (sALT), aspartate aminotransferase (sAST) all obviously raise with CYP2E1 (AH) (P all＜0.05), show that (table 1) appears in hepatic injury.Histopathologic examination as seen, Kupffer hyperplasia hypertrophy, ballooning degeneration and acidophilic necrosis appear in hepatocyte, the downright bad companion of the kitchen range shape that as seen is dispersed in lobule inflammatory cell infiltration.
Compare with the isoniazid group with single, isoniazid and tiopronin compositions group rat sALT, sAST and CYP2E1 (AH) activity descend 35%, 16%, 6% and 17% respectively (P equal＜0.05).Isoniazid and tiopronin compositions group 1 (mass ratio of isoniazid and tiopronin is 5:1) are carried out histopathologic examination, though the visible Kupffer hyperplasia of rat liver, but the point-like necrosis region do not occur, shown that isoniazid and tiopronin compositions obviously reduce (Fig. 4) than isoniazid group liver toxicity.
The comparative study .n of table 1 tiopronin and isoniazid compositions liver toxicity and isoniazid hepatotoxic effect (routine number)=6, x ± s (mean ± standard deviation).
Annotate: compare with matched group,
1)P＜0.05; Compare with the isoniazid group,
2)P＜0.05.
2.2 DNA oxidative damage due to the tiopronin protection isoniazid
CYP2E1 have strong nadph oxidase activity can by mediation substrate reaction of formation oxide (reactive oxygen species, ROS) or free radical.But any molecule of free radical attack cells is wherein to cause DNA damage the most important.The free-radical induced DNA damage includes various ways, as single-strand break, and double-strand break, base modification etc.The DNA base is subjected to free radical and attacks and can form twenties kinds of modified bases, and wherein 8-hydroxyl deoxyguanosine (8-OH-dG) chemical property is stable, and studies show that in a large number 8-OH-dG can be used as the endogenous and extrinsic factor biomarker to the DNA oxidative damage.
Accurate liver slice has been preserved more complete organizational structure and iuntercellular contact, is a kind of and the more identical liver external model of liver internal metabolism.So the accurate liver slice technology of this experiment utilization, observed behind isoniazid or its metabolite generation free radical damage, and compared isoniazid and tiopronin and share influence the DNA oxidative damage to DNA.
The result as seen, isoniazid 0.36mol/L can obviously increase 8-OH-dG growing amount (P＜0.05), shows that this dosage isoniazid causes hepatocyte.On the contrary, isoniazid and tiopronin combination group (get isoniazid and tiopronin crude drug configuration solution, wherein the isoniazid final concentration is 0.36mol/L, and the tiopronin final concentration is 0.072mol/L) are compared with matched group, and DNA base modification thing does not have obvious increase; Compare 8-OH-dG growing amount decline 56% (table 2) with the isoniazid group.
Table 2 tiopronin is obviously protected liver slice DNA oxidative damage .n (routine number)=3 due to the isoniazid, x ± s (mean ± standard deviation).
Annotate: compare with matched group,
1)P＜0.05; Compare with the isoniazid group,
2)P＜0.05.
The prompting of above experimental result, tiopronin can effectively suppress the inducing action of isoniazid to CYP2E1, reduces the free radical that generates through CYP2E1, thus protection DNA avoids oxidative damage, the hepar damnification due to the treatment isoniazid.
3 methods
3.1 the enzyme kinetics parametric measurement of AH
The sacrificed by decapitation rat, the preparation hepatomicrosome is a standard with the bovine serum albumin, presses the Lowry method and measures protein content.The cumulative volume of AH reaction system is 1mL, contains 10mM MgC12,150mM KCl, 50mM Tris-HCl (pH 7.4), 0.8mM nicotinamide-adenine dinucleotide phosphate (NADP), 40mM 1-Hydroxy-1,2,3-propanetricarboxylic acid., 1.2U Isocitrate dehydrogenase, 8mM aniline and 1mg microsomal protein.Reaction system is in 37 ℃ of incubations, and response time 30min measures 630nm place absworption peak.
When measuring the enzyme kinetics parameter, add the aniline of variable concentrations in reaction system, final concentration is respectively 4,16,80,400,800 μ mol/L, and 37 ℃ of incubations, response time 30min is through Lieweaver-Burk double-reciprocal plot method calculating K
mValue and V
MaxValue.
3.2 suppressing kinetic parameter, tiopronin measures
The sacrificed by decapitation normal rat, the preparation hepatomicrosome is a standard with the bovine serum albumin, presses the Lowry method and measures protein content.In external incubation system, aniline final concentration 0.04mmol/L adds tiopronin in the reaction system that microsomal protein concentration is 1mg/mL, and parallel pipe is set.Add the tiopronin final concentration and be respectively 0,0.04,0.08,0.16,0.32,0.48mmol/L, cumulative volume 1mL are in 37 ℃ of incubations, and response time 30min uses the semilog linear regression analysis to calculate IC
50Value.IC
50Be meant and certain enzyme activity can be suppressed 50% required inhibitor concentration.
3.3 tiopronin to isoniazid the experiment in vitro research of inductive high CYP2E1 activity influence
In the external incubation system, microsomal protein final concentration 1mg/mL, aniline final concentration 0.8mmol/L, the adding tiopronin (final concentration is 0,0.01,0.1,0.25,0.5mmol/L), 37 ℃ of incubations, response time 30min measures the AH activity.
3.4 tiopronin is to the experimentation of DNA oxidative damage influence due to the isoniazid
1. the preparation of accurate liver slice, cultivation and drug treating
Normal rat common carotid artery sacrificed by exsanguination, aseptic condition are taken out liver down, move in advance with in oxygen-saturated 4 ℃ of KH (Krebs-Henseleit, pH 7.4) buffer.Cut 10mm * 10mm hepatic tissue blocking, place the microtome specimen groove that contains 4 ℃ of KH buffer, cut into slices (thickness 300 μ m) continue logical oxygen in the overall process.Choose the whole livers section, place the pre-1h of cultivation of DMEM (Dulbcco ' s Modifed Eagle Medium) complete culture solution.
After treating that pre-cultivation finishes, the section of isoniazid group moves into to contain in isoniazid (final concentration is 0.36mol/L) the DMEM complete culture solution cultivates 2h.Isoniazid and tiopronin compositions group (mass ratio of isoniazid and tiopronin is 5:1) section, place the DMEM complete culture solution that contains isoniazid and tiopronin to cultivate 2h and (get isoniazid and tiopronin crude drug, configuration solution, wherein the isoniazid final concentration is 0.36mol/L, the tiopronin final concentration is 0.072mol/L), cultivate 2h.The section of blank group then still places the DMEM complete culture solution to continue to cultivate 2h.Cultivate and finish, take out each group section, through KH buffer rinsing 2～3 times.
2. DNA extraction and digestion
Each group section adds TE buffer 0.4mL homogenate, goes in the Ep pipe, adds 37 ℃ of digestion of cell pyrolysis liquid (containing E.C. 3.4.21.64 60U, RNase A0.06mg) and spends the night, and changes 50 ℃ of vibration digestion 3h again.After treating that sample is cooled to room temperature, with balance phenol extracting 2 times.Get supernatant, add chloroform/isoamyl alcohol (24:1) 0.45mL mixing, through the centrifugal 10min of 5000g.Get supernatant, add 1/10 volume NaAC and 2.5 times of volume dehydrated alcohol, in-20 ℃ of deposit D NA1h, again through the centrifugal 15min of 10000g.Precipitate 2 times with 75% washing with alcohol, vacuum is drained, dissolving DNA.The absorbance of spectrophotometry 260nm and 280nm (A), its A260/A280 are about 2.0, calculate dna content.Add DNaseI (RNase Free) 50U by per 60 μ gDNA, ALP 1.2U and phosphodiesterase 1.2U spend the night in 37 ℃ of digestion.Use the trichloroacetic acid stopped reaction, behind the centrifugal 6min of 12000g, get supernatant and add the NaOH neutralization, use the residual organic facies of water saturation extracted with diethyl ether 2 times ,-20 ℃ frozen.
3. HPLC-MS/MS measures DNA oxidative damage mark 8-hydroxyl deoxyguanosine (8-OH-dG)
Get 10 μ L sample sample introduction analyses, mobile phase: 20mmol/L ammonium acetate (regulating pH to 6.5)-methanol (90:10) with the 1mol/L glacial acetic acid; Flow velocity: 0.2mL/min; Column temperature: 25 ℃; Each sample analysis time spent 20min.Use electron spray ionisation (Electrospray Ionization, ESI), the cation scan mode adopts multiple-reaction monitoring (MRM) mode detection, select many ion pair is carried out qualitative, quantitative.Electron spray voltage is 5200V, 400 ℃ of ion source temperatures.Atomization gas (NEB), gas curtain gas (CUR), collision gas (CAD) and secondary air all use nitrogen, and parameter is respectively NEB 7Psi, CUR 9Psi, CAD 7Psi, secondary air 8L/min.
8-OH-dG, molecular weight are 283.2, under the cation detection mode, produce [M+H] of quasi-molecular ion m/z 284.3 in the Q1 scanning
+The peak carries out daughter ion scanning to it, produces stronger m/z 168.4 fragment ions and is used for quantitatively (Fig. 5 A), uses m/z 140.2 fragment ions auxiliary qualitative simultaneously; In addition, also produce quasi-molecular ion m/z 306.1 [M+Na] in the Q1 scanning
+The peak carries out daughter ion scanning to it, produces stronger m/z 190.2 fragment ions (Fig. 5 B), and m/z 262.1 fragment ions all are used for auxiliary qualitative.DG, molecular weight are 267.2, obtain respectively under the cation detection mode [M+H]
+The quasi-molecular ion peak of (m/z 268.1) carries out daughter ion scanning to it, and the fragment ion that produces m/z 152.2 is used for quantitatively, and it is qualitative to use m/z117.2,135.3,110.4 to assist simultaneously.
With the 8-OH-dG peak height dG peak height is carried out quantitatively, the mass spectrum sketch map is seen Fig. 6.The method lowest detection is limited to 1ng/mL (S/N=3), and relative standard deviation (RSD) is 3.43%.
Claims (10)
1, a kind of pharmaceutical composition contains isoniazid, tiopronin or its sodium salt and pharmaceutically suitable carrier and/or excipient as active component; Wherein the mass ratio of isoniazid and tiopronin is 1～10:1.
2, pharmaceutical composition according to claim 1 is characterized in that: the mass ratio of isoniazid and tiopronin is 5:1.
3, pharmaceutical composition according to claim 1 and 2 is characterized in that: the content of isoniazid is 50～300mg.
4, pharmaceutical composition according to claim 1 and 2 is characterized in that: pharmaceutically suitable carrier and/or excipient are selected from one or more in carboxymethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, maltose, sucrose, medical starch, magnesium stearate, water for injection, antioxidant, the osmotic pressure regulator.
5, pharmaceutical composition according to claim 4 is characterized in that: antioxidant is selected from one or more in sodium sulfite, sodium sulfite, sodium pyrosulfite, ascorbic acid, cysteine hydrochloride, glycine, propylene glycol, the thiourea.
6, pharmaceutical composition according to claim 4 is characterized in that: osmotic pressure regulator is selected from one or more in sodium chloride, glucose, mannitol, xylitol, the potassium chloride.
7, pharmaceutical composition according to claim 1 and 2 is characterized in that: the content of isoniazid is 0.01～10%, and the content of tiopronin is 0.01～2%, and the content of pharmaceutical carrier and/or excipient is 88～99%, and above percentage ratio is mass ratio.
8, pharmaceutical composition according to claim 1 and 2 is characterized in that: described medicine is injection or oral formulations.
9, pharmaceutical composition according to claim 8 is characterized in that: described medicine is sterile powder injection, lyophilized injectable powder, aqueous injection, tablet, capsule, oral liquid, sustained-release preparation or granule.
10, claim 1 or the 2 described pharmaceutical compositions application in preparation treatment tuberculosis.
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| CN109157506A (en) * | 2018-09-12 | 2019-01-08 | 遂成药业股份有限公司 | A kind of isoniazid oral solution and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109157506A (en) * | 2018-09-12 | 2019-01-08 | 遂成药业股份有限公司 | A kind of isoniazid oral solution and preparation method thereof |
| CN109157506B (en) * | 2018-09-12 | 2021-10-08 | 遂成药业股份有限公司 | Isoniazid oral liquid and preparation method thereof |
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