Maraviroc is an orally available antagonist of the CCR5 chemokine receptor,
which acts as a human immunodeficiency virus type 1 (HIV-1) coreceptor. Binding of maraviroc
to this receptor blocks HIV-1 attachment to the coreceptor and prevents HIV-1
from entering host cells.Maraviroc does not require intracellular processing to exert
this activity. Drug interaction studies have shown changes in maraviroc exposure when
given with other anti-HIV medications, and thus quantification of maraviroc in human
plasma is important to manage drug interactions and to evaluate the relationship between
plasma concentrations and treatment response. We developed a conventional LCMS
method for determining plasma maraviroc concentrations, validated by estimating
precision and accuracy for inter- and intraday analysis in the concentration range of
0.011-2.188g/ml. The calibration curve was linear within this range. The average accuracy
ranged from 92.7% to 99.7%, while the relative standard deviations of both interand
intraday assays were less than 7.1%. Recovery of maraviroc exceeded 86.7%. Our LCMS
method provides a conventional, accurate and precise way to determine the maraviroc
concentration in human plasma. This method enables dose adjustment based on monitoring
plasma maraviroc concentrations and permits management of drug interactions
and toxicity

Hirano, Atsushi

Kaneda, Tsuguhiro

Kinoshita, Eri

Nomura, Toshiharu

Okubo, Nami

Takahashi, Masaaki

Tokushima University Institutional Repository

INTRODUCTIONHuman immunodeficiency virus type 1 (HIV-1)treatment has made significant progress with thesuccess of highly active antiretroviral therapy(HAART) using protease inhibitors and reverse tran-scriptase inhibitors. However, long-term use ofHAART is currently limited by developed resistance(1-4) and toxicities (5, 6) associated with many ofthese treatments. New antiretroviral drugs withnovel mechanisms of action and/or distinct HIV-1resistance profiles are thus required to continue ef-fective HAART for the treatment of HIV-1.Maraviroc is one of a new class of antiretroviralcompounds known as CCR5 antagonists. Maravirocprevents HIV-1 from entering CCR5+ CD4+ cells byacting as an antagonist at the CCR5 coreceptor (7-10). This drug in combination with other antiretrovi-ral drugs has been demonstrated to be useful fortreating therapy-experienced and -naïve patientsORIGINALDevelopment and application of a simple LC-MS methodfor the determination of plasma maraviroc concentra-tionsMasaaki Takahashi1,2, Atsushi Hirano1,2, Nami Okubo1, Eri Kinoshita1,Toshiharu Nomura1 and Tsuguhiro Kaneda31Department of Pharmacy and 2Clinical Research Center, National Hospital Organization NagoyaMedical Center, Aichi, Japan ; and 3College of Pharmacy, Kinjo Gakuin University, Aichi, JapanAbstract : Maraviroc is an orally available antagonist of the CCR5 chemokine receptor,which acts as a human immunodeficiency virus type 1 (HIV-1) coreceptor. Binding of ma-raviroc to this receptor blocks HIV-1 attachment to the coreceptor and prevents HIV-1from entering host cells. Maraviroc does not require intracellular processing to exertthis activity. Drug interaction studies have shown changes in maraviroc exposure whengiven with other anti-HIV medications, and thus quantification of maraviroc in humanplasma is important to manage drug interactions and to evaluate the relationship be-tween plasma concentrations and treatment response. We developed a conventional LC-MS method for determining plasma maraviroc concentrations, validated by estimatingprecision and accuracy for inter- and intraday analysis in the concentration range of0.011-2.188g/ml. The calibration curve was linear within this range. The average accu-racy ranged from 92.7% to 99.7%, while the relative standard deviations of both inter-and intraday assays were less than 7.1%. Recovery of maraviroc exceeded 86.7%. Our LC-MS method provides a conventional, accurate and precise way to determine the maravi-roc concentration in human plasma. This method enables dose adjustment based on moni-toring plasma maraviroc concentrations and permits management of drug interactionsand toxicity. J. Med. Invest. 57 : 245-250, August, 2010Keywords : maraviroc ; CCR5 antagonist, HIV-1, LC-MS, therapeutic drug monitoringReceived for publication December 12, 2009 ; accepted March28, 2010.Address correspondence and reprint requests to MasaakiTakahashi, Ph.D., Department of Pharmacy and Clinical ResearchCenter, National Hospital Organization Nagoya Medical Center,4 -1 -1 Sannomaru, Naka-ku, Nagoya, Aichi 460-0001, Japan andFax : +81-52-971-0776.The Journal of Medical Investigation Vol. 57 2010245infected with CCR5 tropic HIV-1 (11, 12).On the other hand, drug interaction studies showchanges in maraviroc exposure when administeredwith other anti-HIV medications, including efavirenzand protease inhibitors (13, 14). These changes re-quire alterations in the doses of maraviroc to use.Therefore, monitoring plasma maraviroc concentra-tions is essential.Fayet et al. (15) and Martin et al. (16) have re-cently determined plasma maraviroc concentrationsusing liquid chromatography-tandem mass spec-trometry (LC-MS/MS). The LC-MS/MS assay isvery sensitive and accurate, but these methods haveseveral disadvantages in terms of cost performanceand essential equipment. Notari et al. (17) also re-ported a determination method using HPLC-UV.However, HPLC method has some complicated pro-cedures, such as the solid-phase drug extraction.In addition, according to our preliminary HPLC ap-plication, the sensitivity of LC-MS methods mustat least be determined for clarification of measure-ments. To bypass these difficulties, we aimed todevelop more conventional procedures for deter-mining maraviroc using LC-MS. The principal ad-vantages of our method are rapid liquid-liquid drugextraction from plasma and use of an available in-ternal standard.MATERIALS AND METHODSChemicalsMaraviroc was supplied by Pfizer (New York,NY, USA) and the internal standard (IS), A-86093(5S,8S,10S,11S)-9-hydroxy-2-cyclopropyl-5-(1-methylethyl)-1-[(2-1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis ( phenylmethyl )-2,4,7,12- tetraaza-tridecan-13-oic acid, 5-thiazolylmethyl ester, wasprovided by Abbott Laboratories (Abbott Park, IL,USA). Their chemical structures are shown inFigure 1. Methanol, hexane, methylene chloride andacetonitrile (Kanto Chemical, Tokyo, Japan) were ofHPLC grade. Ammonium acetate, EDTA and ace-tic acid were purchased from Katayama Chemical(Osaka, Japan). Water was deionized and osmosedusing a Milli-Q system (Millipore, Bedford, MA,USA). All other chemicals and solvents were of ana-lytical grade.EquipmentA Waters Alliance 2695 HPLC system and aMicromass ZQ-2000 MS system (Waters, Milford,MA, USA), controlled with MassLynx version 4.0software (Waters), were used for detection. The ana-lytical column was a SunFire C18 column (3.5 μm,2.150 mm ; Waters), protected by a SunFire C18Guard column.Chromatographic and Mass Spectrometric Condi-tionsThe mobile phase was a mixture of 0.1 mMEDTA in 0.1% acetic acid (A), 100% acetonitrile (B)and 100% methanol (C). An isocratic mobile phaseconsisting of A-B-C (87 : 8 : 5) was used during thefirst 2 min of the run, followed by a linear gradientelution consisting of A-B-C (15 : 80 : 5) for the next8 min. The final conditions (15 : 80 : 5) were main-tained for the final 5 min. The system was then re-equilibrated for an additional 25 min using the in-itial conditions. The flow rate of the mobile phasewas 0.2 ml/min, column temperature was 40, andthe amount of injected sample was 5 μl.The mass spectrometer was operated in positiveion electrospray mode. Capillary sprayer voltage was3.5 kV and sample cone voltage was 30 V for bothmaraviroc and A-86093. The source temperaturewas 120and the desolvation temperature was350. The desolvation and cone gas flow rates wereset to 600 and 50 L/h, respectively. The acquisitionmass range was m/z 200-800 at 0.5 s/scan with a0.1-s interscan delay. All mass spectra were ac-quired in centroid mode.Fig. 1. Chemical structures of maraviroc and the internal standard A-86093.M. Takahashi, et al. Determination of maraviroc concentrations246Quantitative analysis, performed in selected-ionrecording (SIR) mode, detected maraviroc at m/z514.7, and the IS (A-86093) at m/z 748.0, all in theform of ions. Quantitation calculations were per-formed using MassLynx version 4.0 analytical soft-ware.Standard SolutionsStock solutions of maraviroc and A-86093 wereprepared by dissolving accurately weighed amountsof each reference compound in water/ethanol (50 :50, v/v) to yield concentrations of 87.5 μg/ml ma-raviroc and 41.0 μg/ml A-86093. These stock solu-tions were stored at -80and thawed on the dayof analysis. The stock solution was diluted in drug-free plasma to yield maraviroc concentrations of0.011, 0.109, 0.438, 1.094 and 2.188 μg/ml.Sample PreparationA 2-ml aliquot of methylene chloride/hexane(50 : 50, v/v) containing the IS (0.164 μg/ml) and0.3 ml of 0.2 M ammonium acetate were added to500 μl plasma sample prepared from peripheral bloodanticoagulated with heparin. The mixture was vor-texed for 5 min and then centrifuged at 3500g for 5min. The upper layer was separated and evaporateddry. This dried material was then dissolved in 50 μlmobile phase solution. Lastly, 5 μl upper solution wasinjected into the LC-MS system. The institutionalreview board of National Hospital OrganizationNagoya Medical Center approved this study andeach subject provided written informed consent.ValidationInter- and intraday precision values using thismethod were estimated by assaying control plasmacontaining 5 different concentrations of maraviroc5 times on the same day and on 3 separate days toobtain the relative standard deviation (RSD). Accu-racy was determined as a percentage of the nomi-nal concentration. To assess absolute recovery ofmaraviroc extracted from plasma, peak area ratiosof analytes to IS were compared with those ob-tained from the mobile phase with the same con-centration. Mean recoveries were determined in trip-licate.RESULTSLC-MS ChromatogramsFigures 2A and 2B show selected-ion recordingchromatograms obtained from a spiked plasma sam-ple containing 0.875 μg/ml maraviroc and 0.164 μg/Fig. 2. Selected- ion recording chromatograms for maraviroc and A-86093.(A,B) Spiked plasma containing 0.875 μg/ml of maraviroc monitored with m/z 514.7 (A) and 0.164 μg/ml of A-86093 (IS) monitoredwith m/z 748.0 (B).(C,D) A blank plasma sample monitored with m/z 514.7 (C) and m/z 748.0 (D).(C) and (D) are the expanded figures of the baselines in (A) and (B), respectively.The Journal of Medical Investigation Vol. 57 August 2010 247ml A-86093 (IS). Under the described chroma-tographic conditions, retention times were 8.00 minfor maraviroc and 13.6 min for A-86093. Figures2C and 2D show chromatograms obtained from ablank plasma sample. Assays performed on drug-free human plasma successfully showed no inter-fering peaks during the examined intervals of reten-tion times. Figure 2D represents an expanded figureof the baseline part of Figure 2B. These peaks didnot affect the quantification of IS. Anticoagulantsheparin and EDTA did not hinder the selected-ionrecording chromatograms for maraviroc and IS.Validation : Linearity, Precision, Accuracy and Re-coveryCalibration curves of maraviroc appeared linearin the concentration range of 0.011-2.188 μg/ml,with a correlation of 1.000.The precision, accuracy, and recovery of our LC-MS method are shown in Table 1. The selectedconcentration of maraviroc covers the expectedplasma concentrations found in patients. RSDs cal-culated for maraviroc in inter- and intraday assaysranged from 3.2% to 7.1%, accuracy from 92.7% to99.7% and recovery from plasma from 86.7% to89.0%. Mean extraction recovery of IS was 87.9%.These results indicate that this method achieves ahigh degree of reproducibility and accuracy.Clinical applicationFigures 3 and 4 show chromatograms of a plasmasample obtained from an HIV-1 infected patient onFig. 3. Selected- ion recording chromatograms of a plasmasample obtained from patient 1.Table 1. Intra- and interday precision and accuracy for maravirocIntraday (n=5) Interday (n=15)Expected(μg/ml)Measured(μg/ml)RSD(%)Measured(μg/ml)RSD(%)Accuracy(%)Recovery(%)0.011 0.0100.001 7.1 0.0100.001 5.5 92.75.1 86.74.20.109 0.1060.007 6.4 0.1050.007 6.8 96.26.6 88.92.90.438 0.4370.024 5.5 0.4230.022 5.1 96.64.9 89.04.41.094 1.0960.048 4.4 1.0910.051 4.6 99.74.6 87.64.32.188 2.1640.070 3.2 2.1220.081 3.8 96.53.7 87.64.6RSD, relative standard deviation. Values indicate meanSD.Fig. 4. Selected- ion recording chromatograms of a plasmasample obtained from patient 2.M. Takahashi, et al. Determination of maraviroc concentrations248maraviroc.The patient 1 was a Japanese man aged 43 yearswith a body weight of 38.0 kg. He had been treatedwith maraviroc, lamivudine, etravirine, darunavir andritonavir for one month. Maraviroc was administeredat 150 mg once daily, because a strong CYP3A4inhibitor, darunavir, was coadministered with ma-raviroc. However, his CD4+ count was still 20/μlwith a viral load of 100,000 copies/ml. Successful vi-rological treatment was not achieved. The maravi-roc plasma concentration, measured at trough, was0.024 μg/ml. In the DHHS guidelines (18), the sug-gested minimum target trough maraviroc concen-tration is more than 0.050 μg/ml. This treatmentfailure may have been due to a low maraviroc troughlevel and thus to elevate the trough level, we pro-posed increasing the maraviroc dose to 300 mgtwice daily.The patient 2 was a Japanese man aged 34 witha body weight of 75.4 kg. He had been treated withmaraviroc, lamivudine, efavirenz, enfuvirtide, ti-pranavir and ritonavir for two weeks. Maravirocwas administered at 600 mg twice daily, because astrong CYP3A4 inducer, efavirenz, was coadminis-tered with maraviroc. At the start of this regimen, hisCD4+ count was 23/μl with a viral load of 220,000copies/ml. After two weeks, his CD4+ count was69/μl with a viral load of 3,500 copies/ml. The ma-raviroc trough plasma concentration was 0.177 and0.133 μg/ml. In this case, the maraviroc troughlevels were more than 0.050 μg/ml. The viral loadhas been decreasing and treatment success is ex-pected in the future. We thus proposed maintain-ing the current daily dose of maraviroc.DISCUSSIONClinical trials of maraviroc have demonstrated po-tent antiviral responses in patients infected withCCR5 tropic HIV-1 who had previously received tri-ple classes of antiretroviral drugs and/or were iden-tified as triple-class resistant (19). Moreover, ma-raviroc has demonstrated a clean safety profile inthese studies and may not have the toxicities andtolerability issues seen with current anti-HIV drugs.Maraviroc has thus become an important compo-nent of combination treatment regimens for therapy-experienced patients infected with CCR5 tropicHIV-1.Maraviroc is a substrate of CYP3A4 and Pgp, andits pharmacokinetics is likely to be modulated byinhibitors and inducers of these enzymes/transport-ers (13, 14). Therefore, a dose adjustment is re-quired when maraviroc is coadministered with thosedrugs. To manage these drug interactions and en-sure optimal drug efficacy, monitoring plasma ma-raviroc concentrations is essential. For this purpose,we developed a method for determining plasma ma-raviroc concentrations using LC-MS. The maraviroccalibration curve was linear within the concentrationrange of 0.011 to 2.188 μg/ml, and average accu-racy ranged from 92.7% to 99.7%. Both inter- andintraday RSDs for maraviroc were less than 7.1%.Recovery of maraviroc ranged from 86.7% to 89.0%.Thus, our newly developed method achieved a highdegree of reproducibility and accuracy. In addition,as plasma concentrations of maraviroc are report-edly within 0.03-0.89 μg/ml when maraviroc is ad-ministered at 300 mg twice daily (13), our methodsuccessfully covers this region with good precisionand accuracy.Our LC-MS method provides a conventional, ac-curate and precise way to determine the maravirocconcentration in human plasma. This method en-ables a dose adjustment based on monitoring plasmamaraviroc concentrations for HIV-1-infected patientsand permits management of drug interactions andtoxicity.ACKNOWLEDGEMENTSThis study was supported in part by a HealthScience Research Grant for Research on HIV/AIDSfrom the Ministry of Health, Labor, and Welfare ofJapan (H13-AIDS-001 and H16-AIDS-002 to TK)and a Grant-in-Aid for Clinical Research from theNational Hospital Organization to MT.REFERENCES1. de Bethune MP, Hertogs K : Screening and se-lecting for optimized antiretroviral drugs : ris-ing to the challenge of drug resistance. CurrMed Res Opin 22 : 2603-2612, 20062. Machouf N, Thomas R, Nguyen VK, Trottier B,Boulassel MR, Wainberg MA, Routy JP : Ef-fects of drug resistance on viral load in patientsfailing antiretroviral therapy. J Med Virol 78 :608-613, 20063. 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Development and application of a simple LC-MS method for the determination of plasma maraviroc concentrations

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Development and application of a simple LC-MS method for the determination of plasma maraviroc concentrations

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