Concentrations of rifampicin in pre-dose samples in patients with pulmonary tuberculosis

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brazjinfectdis2019;23(2):130–133

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Concentrations

of

rifampicin

in

pre-dose

samples

in

patients

with

pulmonary

tuberculosis

Breno

Kristoffer

Beleza

Uchoa,

Carlos

Augusto

Abreu

Albério,

Ana

Carla

Godinho

Pinto,

Stefania

de

Medeiros

Araujo

Lucena,

José

Luiz

Fernandes

Vieira

∗

UniversidadeFederaldoPará,FaculdadedeFarmácia,LaboratóriodeToxicologia,Belém,PA,Brazil

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o

Articlehistory:

Received27January2019 Accepted2May2019 Availableonline22May2019

Keywords: Infectiousdisease Tuberculosis Rifampicin

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Rifampicinisusedinbothphasesoftreatmentfortuberculosis.Inchronicuse,theshort half-lifeandtheself-inductionofmetabolismcandecreasethelevelsofthedrugbelowthe minimalinhibitoryconcentration.Theaimofthestudywastoinvestigatewhetherplasma concentrationsofrifampicinaresustainedabove0.5␮g/mLinpatientswithtuberculosis using600mg/day.Rifampicinwasmeasuredinplasmabyhigh-performanceliquid chro-matographyandasputumsmearmicroscopywasperformedinalldaysofthestudy.Atotal of44malepatientscompletedthestudy.Ondays31,61and91,themeanplasma concen-trationsofrifampicinwere0.6(0.5)␮g/mL,0.55(0.5)␮g/mLand0.46(0.4)␮g/mL.Therewas ahighvariationofrifampicinlevelsleadingtoahighpercentageofsampleswith concen-trationsbelow0.5␮g/mL.Therewasnosigniﬁcantassociationbetweenthefrequencyof sampleswithdruglevelsbelow0.5␮g/mLwiththeconversionofthesputummicroscopy. Thesedatasuggestthatpre-dosessamplesofferlimitedinformationontheexposureofM. tuberculosistorifampicin.

Tuberculosisis arelevanthealthissuein Brazilwithabout 70,000newcasesdiagnosedeachyear.Thetreatment com-prises a ﬁxed-dose combination of rifampicin, isoniazid, pyrazinamide,andethambutol(RHZE)intheintensivephase andrifampicinwithisoniazid(RH)inthecontinuationphase. RifampicinisahydrazoneofarifamycinBderivativewith N-amino-N-methylpiperazineapprovedbytheFoodandDrug Administrationfortreatmentofthediseasesince1971.1–3

ThetimeofexposureofM.tuberculosistorifampicin con-centrationsabove the MIC (0.25–0.5␮g/mLfor wild-type of

∗ _{Corresponding}_author.

E-mailaddress:jvieira@ufpa.br(J.L.Vieira).

Mycobacterium)isthebestdeterminantofpatientoutcome.4,5

However, there is a high variation in rifampicin plasma concentrations after therapeutic doses, which is related to the patients (age, sex, weight, ethnicity, malabsorption, comorbidity, compliance with prescribed regimen, genetic polymorphism of drug metabolizing enzymes and trans-porters),aswellastothedrugadministered(qualityofthe drugdispensedanddrugformulation).Asaresult,adequate exposureofM.tuberculosistothedrugpromotingdetrimental impactonoutcomecanbeimpaired,suchastreatmentfailure, relapseofinfectionandemergenceofresistance.3,6–8

Chronicuseofrifampicinincreasestherateof disappear-anceofthedrugfrombloodintheearlyphaseofthetreatment

https://doi.org/10.1016/j.bjid.2019.05.001

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brazj infect dis.2019;23(2):130–133

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with a consequent reduction of its half-life as well as of drugexposure,whichisrelatedtoitsshorthalf-lifeand self-inductionofmetabolism.3,9_The_objective_of_the_current_study

wastoinvestigatewhethertheconcentrationsofrifampicin inplasmaaresustainedabovetheMICinpre-dosesamples collected inbothphases oftreatment inBrazilianpatients withpulmonarytuberculosisonchronicuseofdailydosesof 600mgofthedrug.

This was a prospective study carried out between Jan-uary2017and July2018incasesofpulmonarytuberculosis receiving care at the Health Unit of Guama District, City ofBelem,PA,Brazil.Theinclusioncriteriawere adultmale patientswithclinical,laboratoryandradiologicaldiagnosisof pulmonarytuberculosis.Exclusion criteriawereuseof anti-tuberculosisdrugsinthelast30days,casesofretreatment, children, and those coinfected with HIV, hepatitis A, B, C orEviruses, drugaddictionincluding ethanolandtobacco, diabetes mellitus,hepatic or renal diseases, or those with ahistoryofallergy toanti-tuberculosisdrugs.Eachpatient receivedadailytotaldose ofrifampicin (600mg),isoniazid (300mg) pyrazinamide(1600mg) and ethambutol (1100mg) for two months (intensive phase), followed by rifampicin (600mg)plusisoniazid(300mg)forfourmonths(continuation phase).10_In_the_intensive_phase,_the_drugs_were_dispensed_as

fixed-dosecombinationcontainingrifampicin(150mg), isoni-azid(75mg),pyrazinamide(400mg),andethambutol(275mg). In the continuation phase, the drugs were dispensed as fixed-dosecombination containingrifampicin (300mg) and isoniazid (150mg). Both formulations were from the same batchand were provided and analyzed byFarmanguinhos (Fiocruz,RiodeJaneiro,Brazil).Theclinicalstaffofthe tuber-culosisprogramfromthebasichealthunitwasresponsiblefor prescribinganddispensingthedrugs.Thepatientsandtheir relativesreceivedspecificinstructionsaboutthe administra-tiononeachdayandwerestronglyrecommendedtocomplete thecourseoftreatment.Patientswererequestedtoreturnfor follow-upmonthlyontwooccasions: inthefirst,theywere clinicallyevaluated,receivedthemonthlydosesofthedrugs andtheresearchteamsupervisedtheadministrationofthe firstmonthlydose.Inasecondoccasion,thepatientswere requestedtoreturntothehealthunit24hafterthedirectly observeddoseand beforethenextdoseforbloodsampling andsputumsmearformicroscopyondays31,61and91.

Rifampicin was measured by high-performance liquid chromatographyusingaFlexar®system(PerkinElmer),after liquid–liquid extraction from plasma, following the proce-dureproposedbyZhouetal.(2010)withmodiﬁcations.11_The

mobile-phasewasacetonitrile:phosphatebufferpH3.0(40:60 v/v) at a flow of 1mL/min and the column was an RP18, 150mm×4.5mm(PerkinElmer)at30◦C.Theeluentwas mon-itoredat254nm.Thelimitofdetectionwas0.01␮g/mLandthe limitofquantificationwas0.02␮g/mL.Themethodwaslinear from0.02␮g/mLto2␮g/mL.Thewithinandthebetweendays variabilitywere12%and16%andthemeanrecoverywas90%. Qualitative data are presented as frequencies of occur-renceandtheconcentrationsofrifampicininplasmaasmean with95%confidenceinterval.Categoricalvariableswere com-paredbychi-squaredtest.One-wayanalysisofvariancewas usedtocomparetheconcentrationsofrifampicininplasma amongdaysofbloodsampling.DatawereanalyzedusingSPSS

3 2 1 0 31 61 91 Day Rif ampicin ( µ g/mL)

Fig.1–Plasmarifampicinconcentrationsinpatients (n=44)withpulmonarytuberculosisusing600mg/day,24h afterasuperviseddose.Thehorizontallineinlefty-axis correspondstoMICof0.5␮g/mL. 100 80 60 40 20 0 31 61 91 P ercentage (%) Days

Fig.2–Percentageofpatientswithresidualrifampicin concentrationsbelow0.5␮g/mL()and0.25␮g/mL( )in thedaysofbloodsampling.

software,release 21(IBMInc,Chicago,IL,EUA).The signiﬁ-cancelevelwas5%.

ThestudyprotocolwasrevisedandapprovedbytheEthics Committee of the Nucleo de Medicina Tropical da Univer-sidade FederaldoPará(Brazil),undernumber1.591.019.All patientsgavewrittenconsentbeforeinclusioninthestudy.

Forty-fourmalepatientscompletedthestudy.Themean age was 28±12 years (range, 21–45 years), and the mean weight was 68.2±19.7kg (range 60–96kg). Sputum smear microscopywasnegativeinall patientsaftercompletionof theintensivephaseandremainednegativeuntilcompletion of treatment. The mean dose of rifampicin administered was9.37mg/kg/day(range8.4–11.8mg/kg/day).Atadmission, there were no measurable concentrations of rifampicin in allbloodsamples.Inthecourseofthetreatment,themean levelsofrifampicinwere0.6␮g/mL(95%CI=0.43–0.78␮g/mL), 0.55␮g/mL (95%CI=0.34–0.75␮g/mL) and 0.46␮g/mL (95%CI=0.32–0.60␮g/mL) on days 31, 61 (phase 1) and 91 (phase 2), respectively (F=0.8409; p=0.4346) (Fig. 1). There wasa23%decreaseinmeanrifampicinlevelsinthecourse oftreatment.Next,rifampicinlevelswereallocatedasbelow orabovetheMIC.Ondays31,61and91,52.2%(n=23),54.5% (n=24),and59%(n=26)ofpatientsshowedplasmalevelsof rifampicinbelow0.5␮g/mL(X2₌_0.192;_p₌_0.9086),_respectively

(Fig.2).Ofthose,38%(n=17),44%(n=19),and42%(n=18)had concentrations of rifampicin below 0.25␮g/mL (X2₌_0.111;

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braz j infect dis.2019;23(2):130–133

sputumsmearsonday31was70%(n=31),and 55%(n=17) had rifampicin plasma levels below 0.5␮g/mL (X2₌_0.129;

p=0.7194). On day 61, all patients showed conversion of sputumsmears.

Time of exposure is more effective than the amount of absorbed drug to assess efﬁcacy of rifampicin.3,8,12 _In

70s, the ratio of pre-dose concentration to MIC was the mostrelevantpharmacodynamicindextoassessrifampicin exposure.12 _Thereafter, _numerous _ways _to _estimate

expo-sure of M. tuberculosis to the drug were reported, such as the measurement of plasma concentration at 2h (Cmax) and6hafterdrugadministrationortheratioof0–24harea undertheconcentration-timecurve(AUC) tothe minimum inhibitoryconcentration(AUC/MIC).3,8,13–15_However,_there_are

limitationswithbothprocedures,ascollectionofserialblood samplesisalaboriousandexpensiveprocess,andisgenerally notfeasibleinclinicalpractice.

Inthestudy,thepre-dosesampleswerechosentoassess drugexposurebasedonthe followingassertions:(a) thera-peuticdosesshould ensureeffectiveplasma levelsfor24h afterrifampicinintake;and(b)thedrugisactivewhenblood concentrationsareabovetheMIC.12,15_Furthermore,_the

sam-plingschedulewasbasedonreportsshowingtheabilityof rifampicintoinduceCYPisoformsismostevidentintheearly phaseofthetreatment.3,6,7,12

All plasma samples collected on D0 were negative for rifampicin,conﬁrmingthatthe drughadnotbeen recently usedbythepatientsenrolledinthestudy.Themeanlevelsof rifampicinwereabovetheMICondays31and61,butahigh percentageofsamplespresenteddruglevelsbelow0.5␮g/mL. Thisﬁndingcanbeduetoboththeshorthalf-lifeofthedrug aswell astothe normalvariationofthe residuallevels of rifampicin.3,6,7,12

Theconcentrations of rifampicin were similar through-outthestudy,withanon-signiﬁcantdecreaseof23%inthe courseofthetreatment,whichcouldbeduetothecapacity ofrifampicintoinduceseveralCYPisoformsandtransporters asMDR1andMDR2.Suchinductionbringsdrug concentra-tion to belowthe MIC and increases the range ofresidual levelsofthedrug.3,16,17 _Furthermore,_several_factors_related

tothepatients,tothedrug,andtothepolicyofdiagnosisand treatmentofthediseasealsoinﬂuencetheconcentrationsof rifampicin.3,6,7,13,14 _Therefore,_rifampicin _levels_in_pre-doses

samplesofferlimitedinformationontheexposureofM. tuber-culosis.

Thefrequencyofsampleswithdruglevelsbelow0.5␮g/mL wasnotassociatedwithconversionofsputummicroscopy, whichcorroboratesthelimitedinformationofdrugexposure inpre-dosessamplesaswellasthedifficultytointerpret clini-calandbacteriologicaloutcomesofasingledruginafour-drug regimen.Furthermore,variablesofM.tuberculosis,asthe var-iousgrowth ratesinvivo,makeitdifficulttocorrelatedrug efficacywithtimeofconcentrationabovetheMIC.3,5,8,12

Inconclusion,thelevelsofrifampicinwerebelowtheMIC inmostofthesamplesinbothphasesoftreatment,andthere was no signiﬁcant association between frequency of sam-pleswithdruglevelsbelow0.5␮g/mLwithconversionofthe sputumsmears.Thesedatasuggestthatpre-dosessamples

offerlimitedinformationontheexposureofM.tuberculosisto rifampicin.

Funding

Thereisnofundingsourcetodeclare.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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