秦瑞玲，徐永吉，吳棟清，王　洋，魯艷柳，陸遠(yuǎn)富

(遵義醫(yī)學(xué)院 基礎(chǔ)藥理教育部重點實驗室暨特色民族藥教育部國際合作聯(lián)合實驗室，貴州 遵義　563099)

Tuberculosis is a chronic and slow-onset infectious disease with an increasing incidence.Tuberculosis patients mainly accept drug treatment.Common drugs are isoniazid,rifampin,streptomycin,pyrazinamide and so on.Isoniazid (INH) as a first-line clinical anti-tuberculosis drug was widely used in clinical practice due to its stable nature,selective effect on mycobacterium tuberculosis,and strong antibacterial effects,with high efficacy,oral administration,and cheap.Isoniazid is a concentration-dependent drug,its plasma concentration determines its bactericidal effect,but high-dose isoniazid has obvious adverse reactions such as hepatotoxicity,and dose-related[1].Clinically,anti-tuberculosis drugs can affect the absorption of vitamins,so doctors recommended that tuberculosis patients ate more foods and fruits vitamins-rich.Because of citrus fruits containing vitamins-rich,tasting good,it is the best choice to patients[2-3].However,citrus fruits can cause many food-drug interactions.There had not been reported that food-drug interactions between citrus fruits and isoniazid.However,it is important to explore the interaction between citrus fruits and anti-tuberculosis drugs for reasonable and safe medication.

1　Materials and methods

1.1Animal studyAll rat experiments were performed in accordance with the Institutional Animal Care and Use Committee at the Ministry of Education Key Laboratory of Basic Pharmacology,Zunyi Medical University,and the Guidance for Ethical Treatment of Laboratory Animals (The Ministry of Science and Technology of China,2006).Male SD rats (180～220 g) were purchased from the Third Military Medical University of Chongqing Animal Center,license number:SCXK (Yu) 2012-0005 (Chongqing,China).Rats were maintained under controlled temperature of (24±2) ℃ and relative humidity of 60%±10% with a 12 h light/dark cycle.Commercial rat chow was available ad libitum except for an overnight fasting period before dosing.All rats had free access to water.

1.2Chemicals and reagentsIsoniazid,acetyl isoniazid and isonicotinic acid were purchased from China Food and Drug Control Institute (100578-200401),Toronto Research Chemicals (Toronto,Canada),China JLG Technology Co.Ltd.(140345),respectively.Paracetamol (as internal standard),isoniazid tablets and orange juice was obtained from China Jinhua Pharmaceutical Co,Ltd.Chengdu,Meilun biological (N1025A),China Huiyuan Juice Group (Huiyuan,100%,1L),respectively.HPLC-methanol and HPLC-acetonitrile were acquired from Shanghai Anpel Scientific Instrument Co,Ltd.(Shanghai,China).Water was prepared in house with Milli-Q water purification system (Millipore,Milford,MA,USA).All other chemicals were of analytical grade.

1.3Experimental protocolThe SD rats were randomly divided into four groups (n=6),isoniazid administration group received isoniazid (dissolved in distilled water) intragastric administration dose of 30 mg/kg at 9∶00 am.Orange juice group was orally administration of 20 ml/kg at 9∶00 am.Orange juice+INH group was orally given isoniazid 30 mg/kg at 9∶00 am after six hours,with a single dose of 20 ml/kg orange juice again.The control group was given the distilled water administration dose of 30 ml/kg at 9∶00 am and 3∶00 pm,respectively.All the group were given continuous administration for 180 days and were given orally isoniazid single dose of 90 mg/kg at the last day.

1.4Sample collectionBlood samples (approximately 0.2 ml) were collected in preheparinized Eppendorf tubes from ether-anesthetized rats just before and at 10,20,30,45,60,75,90,120,150 and 180 min following INH administration.The obtained plasma was stored at -80℃ until analysis.Blood samples were analyzed by LC-MS.At the same time,gene expression of transporters in duodenum and kidney were examined by RT-PCR.The metabolizing enzymes in liver which played key roles in metabolism of INH were examined by Western-blot.

1.5LC-MS method for determination of isoniazid in plasma and isoniazid,acetyl isoniazid and isonicotinic acid concentrations in liver tissues

1.5.1LC-MS conditionsThe LC-MS is composed of Agilent 1100 liquid chromatography system ,equipped with a binary pump,a vacuum degasser unit,an autosampler and an API 3 200 triple quadrupole mass spectrometer with an ESI source.The chromatographic separation was achieved on Angilent reversed phase C18 column(250 mm×4.6 mm,3.5 μm),and the column temperature was set at 40 ℃.The mobile phase was consisted of acetonitrile (B) and 0.1% ammonium aqueous solution (A) using gradient elution of 5% (B) at 0～1 min ,5%～95% (B) 2～6 min,95% (B) at 6～8 min,5% (B) at 8～10 min,and re-equilibration time of gradient elution was 4 min.The flow rate set at 0.3 ml/min.The sample injection volume was 5 μl.All data acquisition and peak integration were performed using Analyst software (version 1.4.2) from Applied Biosystem/MDS Sciex.The analytes were determined in positive ionization mode with isoniazid (Mr:137.06),acetyl isoniazid (Mr:180.08),isonicotinic acid (Mr:123.10),proton nuclear ratio:isoniazid (138.00 ～138.50),acetyl isoniazid (180.00～180.50),isonicotinic acid quantified (124.00 ～124.50) by multiple-reaction monitoring (MRM) mode.Mass spectrometry was operated with an optimized spray voltage at -4 500 V,turbo spray temperature at 400 ℃,and collision gas at 5 psi.The curtain gas,nebulizer gas (gas 1) and auxiliary gas (gas 2) were at 30,60 and 60 psi,respectively.

1.5.2Preparation of isoniazid,acetyl isoniazid and isonicotinic acid standard and quality samplesTwo sets of isoniazid,acetyl isoniazid and isonicotinic acid stock solutions at 1 mg/ml were prepared in methanol with separately weighed isoniazid,acetyl isoniazid and isonicotinic acid,respectively.One solution was used to spiked standards and the other for quality control (QC) samples.Calibration standard concentrations of isoniazid,acetyl isoniazid and isonicotinic acid 0.025～0.3,0.1～0.6,0.05～0.6 μg/ml,respectively.They were prepared by spiking isoniazid,acetyl isoniazid and isonicotinic acid stock solutions into blank human plasma.Paracetamol was used as internal standard and prepared with 20% methanol at 6 μg/ml in working solution.The stock solutions,standards,QC samples and the internal standard solution were stored at -20℃ between uses.

1.5.3Plasma sample preparationTo 20 μl aliquot of each standard,QC sample,blank plasma was added 18 μl internal standard (6 μg/ml paracetamol).After briefly mixing on a vortex shaker for 30 s,the sample were deproteinized by adding 362 μl methanol and vortex-mixing for 30 s.Then samples were centrifuged at 14 000gfor 15 min.A 200 μl of the clear supernatant was transferred into autosampler vial and 5 μl was injected into the LC-MS system.All plasma samples needed to be processed immediately once they were thawed at room temperature.

1.6LC-MS method for determination of isoniazid,acetyl isoniazid and isonicotinic acid in liver tissuesAn LC-MS method for the determination of isonazid,acetyl isoniazid and isonicotinic acid in rats liver tissues was developed and validated following the determination of isoniazid in rat plasma.

1.6.1Preparation of isoniazid,acetyl isoniazid and isonicotinic acid standard and quality samplesAn LC-MS method for the determination of isonazid,acetyl isoniazid and isonicotinic acid in rat liver tissues was developed and validated following the determination of isoniazid in rat plasma.

1.6.2Liver tissue samples preparationWeighing 100 mg of liver tissue samples and adding 100 μl PBS buffer solution,the mixture was homogenized and centrifuged at 10 000 g for 10 min.Then 80 μl of supernatant was mixed with 300 μl methanol and 50 μl of internal standard working solution and the mixture was vortexed for 30s.After centrifugation at 14 000gfor 15 min,200 μl of supernatant was transferred into a sample vial.Subsequently,5 μl of the mixture was injected into the LC-MS system.

1.7Method validation procedure

1.7.1Calibration curveCalibration curves were obtained by linear regression of the peak area ratio of isoniazid,acetyl isoniazid and isonicotinic acid to internal standard versus the nominal isoniazid,acetyl isoniazid and isonicotinic acid concentrations .

1.7.2Lower limit of quantification (LLOQ)The LLOQ was established using six samples independent of standards to determine accuracy and precision.The accuracy should be within 20% of the nominal concentration and precision should be <20%.

1.7.3Intra-day and inter-day precision and accuracyIntra-day precision and accuracy were determined by analysis of six replicates of each QC sample:isoniazid,acetyl isoniazid and isonicotinic acid (n=6),at low (0.05,0.1,0.05 μg/ml)，medium (0.2,0.4,0.2 μg/ml),and high (0.45,0.45,0.375 μg/ml) concentration levels extracted with a set of standards in one batch,respectively.The same procedure was repeated on five different days with new samples to determine inter-day precision and accuracy.Precision was reported as RSD and accuracy as percent of the nominal concentration (% deviation).

1.7.4RecoveryThe recovery of isoniazid,acetyl isoniazid and isonicotinic acid from the blank plasma following sample preparation was assessed by comparing the peak area of isoniazid,acetyl isoniazid and isonicotinic from plasma extracts to the area of the same concentration of isoniazid,acetyl isoniazid and isonicotinic acid spiked into blank plasma extracts after precipitation.

1.7.5StabilityThe stability of isoniazid,acetyl isoniazid and isonicotinic acid in rat plasma was evaluated at the conditions:2 freeze-thaw cycles,stored at -20 ℃ for 27 d,room temperature (22 ℃) for 1,3 and 20 h.Each condition was tested with QC samples at low and high concentration levels in triplicated.Fresh samples were used as reference.Stability of processed samples was evaluated after standing on the bench for 3 days.

1.8Total RNA isolation and real-time RT-PCR analysisTotal RNA was isolated from Trizol and total RNA was detected by UV spectrophotometer.The quality of RNA was confirmed by the ratio of 260/280 (1.8～2.0) before further experiments.The total RNA concentration was adjusted to 100 ng/μl reverse transcription (37 ℃,15 min; 85 ℃,5 s),the resulting cDNA was used for SYBR Green PCR (annealing temperature 60 ℃),β-actin as an internal reference using specific primers such as Table 3.

Tab1PrimersequencesusedforRT-PCRreaction

Gene NameGenBank NumberForward primer (5' - 3')Reverse prime (5' - 3')β-actinNM_007393catccgtaaagacctctatgccaacatggagc-caccgatc-cacaBCRPNM_181381ccagcctcggtattccatctcagc-cgaagaatctc-cgttgATP8B1NM_001106140atggccctggaaatcacagacggtgttc-ctaatcacg-cagMRP 4NM_001105515cttggagaggagttgcaagggctgtgt-tcaaagc-cacagaMATE2-KNM_001191920cacctcccagttcttcctgttc-ccaatctc-gaaggtc-cac

1.9Western-blot detection of liver INH metabolism-related protein expression Taking 100 mg of liver tissue,adding 1 mL of RIPA lysate and 10 μl of PMSF,homogenate and centrifuge the supernatant to determine the protein concentration by BCA protein quantitative kit.Add 4 × protein loading buffer to denature at 95 ℃.10% Bis-Tris gel electrophoresis,the use of PVDF membrane protein transfer membrane,after blocking,incubated at 4 ℃ overnight,incubated in TBST buffer incubated secondary antibody 1 h.Then the membrane was added drop-wise a chem luminescence liquid exposure and development.

2　Results

2.1LC-MS detection of plasma isoniazidTo establish a method for the determination of isoniazid and its major metabolites acetyl isoniazid and isonicotinic acid in rat plasma by ultra performance liquid chromatography-mass spectrometry (1.5.3 LC-MS method):Under this chromatographic conditions,hydrazine,isoniazid and isonicotinic acid in the chromatographic conditions phase response signal obvious,good shape,with good separation of other impurities.

2.2LC-MS detecting plasma samples of drug concentrationsAfter continuous administration of 180 d in each group,given isoniazid,measure isoniazid plasma drug concentration and time curve:compared with the control group,after each group taking the orange juice 180 days,isoniazid plasma drug concentration in the 10～90 min period was significantly higher than the blank group,shown in Fig 1; compared with isoniazid alone,the isoniazid plasma concentration was significantly higher than orange juice+INH for 180 days,as shown in Fig 2,with a statistically significant difference (P<0.05,n=6).

*P<0.05,vs control group,n=6.　　Fig 1　Changes in plasma concentration-time curve of INH in rat plasma samples between blank group and orange juice group which were treated with oral administration for 180 d

*P<0.05,vs INH group,n=6.　　Fig 2　Changes in plasma concentration-time curve of INH in rat plasma samples between INH group and orange juice combined INH group which were treated with oral administration for 180 d

2.3LC-MS detection of liver tissues drug concentrationsAfter continuous administration for 180 days,the isoniazid and its metabolites acetyl isoniazid and isonicotinic acid concentrations in liver are shown in Fig 3.The concentrations in orange juice group and the orange juice+INH group concentrations were significantly higher than the blank group.Every group were significantly high concentrations of acethyl isoniazid (P<0.05,n=6).Compared with the isoniazid group,the concentrations in orange juice group and orange juice+isoniazid group were significantly increased.The difference was statistically significant (P<0.05,n=6).

vs control group,*P <0.05; vs INH group,# P<0.05,n=6.　　Fig 3　Changes in the contents of INH,AcINH and INA in liver at 210 min among the four groups which were treated with oral administration for 180 d

2.4RT-PCR method to detect intestinal and renal transporter mRNA relative expression

2.4.1Intestinal transportersCompared with the control group,the orange juice group and the orange juice+INH group inhibited the expression of BCRP and ATP8B1 mRNA,while the isoniazid group decreased the expression of BCRP mRNA (Fig 4).

vs control group,*P <0.05,n=6.　　Fig 4　The mRNA expression of secretion transporters BCRP,ATP8B1 in duodenum among the four groups which were treated with oarl administration for 180 d

2.4.2Kidney transportersThe mRNA expression of renal transporters was detected by RT-PCR method,as shown in Fig 5,orange juice inhibit renal efflux transporter MRP4,MATE2-K mRNA relative expression.Compared with the control group,the expressions of BCRP,MRP4 and MATE2-K mRNA of the orange juice group and the isoniazid group were decreased,while the relative expression of MRP4 mRNA increased in the isoniazid group (P<0.05,n=6).

vs control group,*P<0.05,n=6.　　Fig 5　The mRNA expression of secretion transporters MRP4,MATE-K in kidney among the four groups which were treated with oral administration for 180 d

2.5Western-blot detection of metabolic enzymes in liverCompared with the control group,the relative expression of NAT2 in the liver tissues of the orange juice group and the orange juice+INH group was significantly increased (Fig 6).FAAH1 and GLS2 protein expression significantly increased in each group,compared with the control group (Fig 6) CYP2E1 protein expression in orange juice+INH group and isoniazid group were increased (P<0.05,n=6),as shown in Fig 6.The expression of GST protein in the liver tissues in orange juice+INH and isoniazid groups were significantly increased,as shown in Fig 6(P<0.05,n=6).

vs control group,*P <0.05; vs INH group,# P<0.05,n=6.　　Fig 6　Changes in protein expression of INH metabolism in liver among the four groups which were treated with oral administration for 180 d

3　Discussion

Food-drug interactions (FDI) can lead to drug absorption decrease or increase excretion[4],and even lead to treatment failure,frequent dose changes,antibiotic resistance and mortality increase[5].Citrus fruits contain a variety of bioactive substances,such as naringin,hesperidin,etc.It can interact with a variety of oral drugs.It has been confirmed in the literature that citrus fruits or their fruit juices can interact with anti-allergic drugs,antibiotics,anti-malarial drugs,anti-anxiety drugs[6]and can increase certain CNS drugs,cardiovascular drugs of drug plasma concentration,enhanced risk of adverse reactions[7].On the contrary,it can decrease some drugs systemic exposure,such as pirfenidone[8],paracetamol,to reduce its drug bioavailability,lowering the therapeutic effect.However,there is no report that citrus fruits have an effect on tuberculosis treatment.Tuberculosis is a chronic and slow infectious disease.With the spread of environmental pollution and AIDS,the incidence of tuberculosis is more and more severe[9-10].Isoniazid as a clinical first-line anti-TB drug,its stable properties,strong antibacterial and oral utilization advantages are widely used in anti-TB treatment.The main limiting factor for INH clinical usage is its hepatotoxicity[11-14],resistance[15-16]; The increasing doses of INH promote the hepatotoxicity and dose reduction may lead to drug resistance[17].In addition,compared with adults,children is more sensitive to the concentration of isoniazid in the body[18]and is more likely to cause adverse effects such as hepatotoxicity[19-20].Maintaining a suitable concentration of isoniazid in the body is essential for tuberculosis treatment.Tuberculosis patients generally need long-term medication,and citrus juice orange juice tastes good,large output,with a high market share[2-3],playing an important role in the patient's recipes,the patient is easy to take isoniazid at the same time drinking orange juice,but the orange juice and isoniazid interaction has not been reported,so the study of orange juice on the interaction of isoniazid is of great significance.

Isoniazid is metabolized by N-acetyltransferase 2 (NAT2) in the liver to Acetyl isoniazid (AcINH) with free hydrazine,which is hydrolyzed by amidase (eg ,fatty acid amide hydrolase FAAH1,glutamine amide hydrolysis (GLS2),generating isonicotinic acid (INA) and acetohydrazide (AcHz); isoniazid in the liver is mainly metabolized by NAT2 enzymes,accounting for 50-90% of the isoniazid metabolism[21].The genetic polymorphism of NAT2 is one of the reasons for INH metabolism in individuals[22-23],an increase in NAT2 expression results in a significant increase in the overall hepatic metabolic rate of isoniazid and a rise in the rate of formation of toxic metabolites of isoniazid[21],affecting isoniazid antimicrobial effects and side effects[23].In addition,isoniazid can also be directly hydrolyzed by amidase to isonicotinic acid and free hydrazine (Hz).Free hydrazine is acetylated by acetylation of NAT2 to acetohydrazide.Free hydrazine and acetohydrazide are toxic metabolites of isoniazid and are partially metabolized directly by NAT2 ,Part of the toxic reaction with the cytochrome CYP2E1[22]is a major factor in the liver toxicity caused by isoniazid[23].The results of this study showed that the combined use of orange juice and isoniazid for 180 days resulted in the increasing of blood concentration of isoniazid.Moreover,taking 180 d of orange juice also caused the increase of isoniazid metabolites such as isoniazid and isonicotinic acid in the liver.Drug transporter is a functional protein located on the cell membrane,playing a decisive role in the drug transport,which affect drugs pharmacokinetics behaviors.The protein expression in the liver was detected by Western-blot.The results showed that NAT2 expression was increased in the orange juice group and the orange juice group and the isoniazid group for 180 days.The expression of FAAH1 and GLS2 protein were increased in the orange juice,orange juice,isoniazid and isoniazid groups,orange juice+INH group and isoniazid group CYP2E1 protein expression increased,as shown in Fig 6.orange juice caused by the increase of isoniazid absorption,but in the drug half of curve isoniazid plasma concentration was no significant difference; The above results confirm that drinking orange juice can increase the expression of NAT2,FAAH1 and GLS2 involved in the metabolism of isoniazid in the liver,causing the overall metabolic rate of isoniazid to increase,which may affect the toxicity of isoniazid[24].

Toxic metabolites produced by isoniazid are catalyzed by GST[24].In this experiment,western blot analysis of liver protein expression found that orange juice or isoniazid induced GST protein expression after 180 days administration (Fig 6).Orange juice can cause the increase of isoniazid absorption,and the overall metabolic rate of isoniazid is accelerated,and the toxic metabolites are increased.After drinking orange juice for 180 days,the effect on GST protein expression is unobvious,so the ability of the body to eliminate the isoniazid toxicity products is unknown.The above results suggest that:Clinically it should be cautious drinking orange juice when ingestion of isoniazid.It also suggests that there may be a food-drug interaction between orange juice and other drugs.

In summary,this manuscript for the first time studied the orange juice and isoniazid food-drug interactions and found that orange juice can affect isoniazid pharmacokinetics after a long treatment in rats,its combination with isoniazid increased plasma isoniazide concentration and the metabolic rate.At the same time,it can be inferred that isoniazid translocates in the gut and the kidneys,which may be involved in transporters BCRP,ATP8B1 and the renal efflux transporters MRP4 and MATE2-K; Long-term drinking orange juice can inhibit the intestinal transporter BCRP,ATP8B1 and renal efflux transporter MATE2-K expression,and renal efflux transporter MRP4 mRNA expression.