한국임상약학회지 제16권 제1호
`Kor. J. Clin. Pharm., Vol. 16, No. 1. 2006
`
`Evaluation of the Bioequivalence of Two Brands of Naltrexone
`50 mg Tablet in Healthy Volunteers
`
`In-hwan Baeka, Hwi-yeol Yuna, Wonku Kangb and Kwang-il Kwona,*
`aCollege of Pharmacy, Chungnam National University, Daejeon, Korea
`bCollege of Pharmacy, Catholic University of Daegu, Kyungbuk, Korea
`
`날트렉손은 µ-opioid 수용체에 특이적이고 선택적으로 길항작용을 나타내어 마약이나 마약성 진통제의 강한 의존성
`치료에 쓰일 뿐만 아니라, 알코올 의존성 치료에도 쓰이는 약물이다. 본 연구는 날트렉손 제제인 레비아 정(50 mg
`tablet, 제일약품)을 대조약으로 하여 시험약인 명인 제약의 트락손 50 mg정의 생물학적 동등성 평가를 하기 위해
`22명의 건강한 지원자를 모집하였다. 지원자를 두 군으로 나누어 1정씩 투여하였고 2×2 교차시험을 실시하였다. 날
`트렉손의 혈장 중의 농도를 정량하기 위하여 발리데이션된 LC/MS/MS를 사용하였다. 채혈 시간은 투약 전 및 투약
`후 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 9, 12 시간에 걸쳐 시행하였다. 생물학적 동등성을 판정하기 위한 파라미
`터로 12시간까지의 혈장 중 농도 곡선 하 면적(AUC12hr)과 최고 혈중 농도(Cmax)를 사용하였다. AUC12hr 의 평균은
`43.45 ng·hr/ml (시험약) 과 43.31 ng·hr/ml (대조약) 으로 관찰 되었고, Cmax의 경우 각각 12.01 ng/ml (시험약) 과
`12.27 ng/ml (대조약) 으로 관찰 되었다. AUC12hr 의 경우 로그변환한 평균치 차의 90%의 신뢰구간이 log0.95 – log1.07
`이었고, Cmax의 경우 log0.87 – log1.14로 계산되어 두 항목 모두 log0.8 – log1.25이어야 한다는 식품의약품 안전
`청과 FDA의 기준을 모두 만족시켰다. 이상의 결과를 종합하면 시험약 트락손 정 50 mg은 대조약 레비아 정 50
`mg에 대하여 생물학적으로 동등한 것으로 판정되었다.
`□ Key words − Naltrexone, bioequivalence, LC/MS/MS, Traxone, Levia®
`
`Bioequivalence of two formulations of the same drug
`
`comprises equivalence with respect to the rate and extent
`
`of their absorption. While the area under concentration
`
`time curve (AUC) generally serve as the characteristic of
`
`the extent of absorption, the peak concentration (Cmax)
`and the time of its occurrence (Tmax), reflect the rate of
`absorption, especially in fast-releasing drug formula-
`tions.1,2) The present study was conducted to evaluate the
`bioequivalence of two brands of naltrexone 50 mg tablets
`
`in fasting, 22 healthy human volunteers. Typical bio-
`
`availability, including AUCt (the area under the plasma
`concentration-time curve from 0 until the last sampling
`
`time, 12 hr) and Cmax (the maximum plasma concentra-
`tion) parameters were compared.
`
`Naltrexone,17-(cyclopropylmethyl)-4,5-epoxy-3,14-
`
`dihydroxymorphinan-6-one, as shown in Fig. 1, has long
`
`Fig. 1. Molecular structure of naltrexone
`
`receptors, with a relative seletivity for the µ-opioid
`receptor at lower doses.3) Naltrexone block the effects of
`
`opioids by competitive binding at opioid recepters. Also,
`
`naltrexone is effective medication for treatment of alco-
`
`hol dependence but the mechanism of action of naltrexone
`
`in alcoholism is not understood but involvement of the
`endogenous opioid system is suggested.4)
`Following oral administration, naltrexone undergoes
`
`been available as an orally available antagonist at opioid
`
`rapid and nearly complete absorption with approximately
`
`Correspondence to : Kwang-il Kwon
`충남대학교 약학대학 임상약학연구실
`대전광역시 유성구 궁동 충남대학교 약학대학
`Tel: +82-42-821-5937, Fax: +82-42-823-6781
`E-mail: kwon@cnu.ac.kr
`
`96% of the dose absorbed from gastrointestinal tract.
`
`Naltrexone is primarily eliminated from the body by
`
`hepatin metabolism and the major metabolite of naltrex-
`
`one is 6-β-naltrexol. The percentage of the administered
`
`dose excreted in urine as free naltrexone is about 1%.
`
`69
`
`AMN1039
`IPR of Patent No. 7,919,499
`
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`70
`
`Kor. J. Clin. Pharm., Vol. 16, No. 1, 2006
`
`Reported half-life for naltrexone, after aral administra-
`tion, is about 4hr.5)
`The purpose of this study was to determine the phar-
`
`and beverages 48 hr before the study, until the last sam-
`pling time.6)
`This study was based on a single-dose, randomized,
`
`macokinetic parameters of two brands of naltrexone 50
`
`two-treatment, two-period crossover design and was
`
`mg capsules and then to compare these parameters statis-
`
`approved by a local ethics committee. All the volunteers
`
`tically to evaluate the bioequivalence between the
`
`signed a written informed consent, in accordance with
`
`brands. Traxone 50mg (Myung In Pharm. Co., Ltd.,
`Seoul, Korea) was used as test product while Levia® 50
`mg (Jeil Pharm. Co., Ltd., Seoul, Korea) was used as ref-
`
`the Korea Guidelines for Bioequivalence Tests (KGBT
`
`1998). In the morning of period ², after an overnight fast-
`
`ing (10 hr) volunteers were given single dose of either
`
`erence product in 22 healthy volunteers.
`
`formulation (reference or test) of naltrexone 50mg with
`
`Materials and Methods
`
`dose administration. Water intake was allowed after 2 hr
`
`240 ml of water. No food was allowed after 4 hr after
`
`Test and reference products
`
`of dose; water, lunch and dinner were given to all volun-
`
`teers according to the time schedule. They were not per-
`
`The test product, Traxone 50 mg (50 mg of naltrexone
`
`mitted to lie down or sleep for the first 4 hr after the
`
`hydroxide, lot no. 353501, Myung In Pharm. Co., Ltd.)
`and the reference product, Levia® 50mg (50 mg naltrex-
`one hydroxide, lots no. RVE301, Jeil Pharm. Co., Ltd.)
`
`dose. Approximately 10ml of blood samples for naltrex-
`
`one assay were drawn into heparinized tubes through
`
`indwelling cannula before(0 hr) and at 0.25, 0.5, 0.75, 1,
`
`were supplied by tablets.
`
`Subjects and methods
`
`1.5, 2, 3, 4, 6, 9, and 12 hr after drug administration. The
`
`blood samples were centrifuged at 3000 rpm for 15 min;
`plasma was separated and kept frozen at -70oC until the
`
`The 50 mg naltrexone bioequivalence study involved
`
`LC/MS/MS analysis. After a washout period of 6 days
`
`22 healthy volunteers with the age from 19 to 28 years
`
`the study was repeated in the same manner to complete
`
`(23.09±2.18 years), in weight from 55 to 96 kg (71.00±
`
`the crossover design.
`
`9.15 kg), and height from 167 to 182 cm (174.77±4.25).
`
`All the volunteers were enrolled after passing a clinical
`
`Chromatographic conditions
`
`examination, including a physical examination and labo-
`
`The plasma naltrexone concentrations were quantified
`
`ratory tests (blood analysis: hemoglobin, hematocrit,
`
`using liquid chromatography-mass spectrometry with a
`
`WBC platelets, WBC differential, blood urea nitrogen,
`
`PE SCIEX API 2000 LC/MS/MS System (Applied
`
`total bilirubin, cholesterol, total protein, albumin, alka-
`
`Biosystems Sciex, Ontario, Canada) equipped with an
`
`line phosphate, glucose fasting, ALT, and AST, and urine
`
`analysis: specific gravity, color, pH, sugar, albumin,
`
`electrospray ionization interface used to generate positive
`ions [M+H]+. The HPLC system was an Agilent 1100
`
`bilirubin, RBC, WBC, and casts). Any with potential
`
`series (Wilmington, DE., USA). The separation was
`
`hypersensitivity to this type of medication, a history of
`
`the hepatic, renal, or cardiovascular disease, or chronic
`
`performed by using a reversed-phase Eclipse XDB-C18
`column (2.1×100 mm internal diameter, 3.5 µm particle
`
`alcohol consumption or other medications was excluded.
`
`This criteria was applied to elimination the source of
`
`variation which can influence the pharmacokinetics of
`
`size; Agilent technology, Wilmington, DE., USA). The
`column oven temperature was set at 30oC. The mobile
`phase consisted of 0.1% formic acid in acetonitrile and
`
`the drug. All the volunteers were restricted not to take
`
`0.1% formic acid in purified water (95: 5% [vol/vol]).
`
`using other drugs from at least one week before the study
`
`The mobile phase was eluted using an aglilent 1100
`
`and until the completion of the study. They also refrained
`
`from alcoholic beverages and xanthine-containing foods
`
`series pump G1312A (Agilent technology, Wilmington,
`DE., USA) at 0.2 ml/min.7)
`
`AMN1039
`IPR of Patent No. 7,919,499
`
`

`

`Evaluation of the Bioequivalence of Two Brands of Naltrexone 50 mg Tablet in Healthy Volunteers
`
`71
`
`A PE SCIEX API2000 triple-stage quadrupole mass
`spectrometer interfaced to a TurboIonSpray® source was
`used for mass analysis and detection. Ionization of
`
`analytes was carried out using the following settings of
`
`the electrospray ionization (ESI) in the positive ion
`mode: TurboIonSpray® temperature, 500oC; ion source
`
`voltage, 5500V; nebulizing gas flow (high-purity air),
`
`1.04 L/min; curtain gas flow (nitrogen), 1.44 L/min; aux-
`
`iliary gas flow, 4.0 L/min; collision gas (nitrogen)
`pressure, 5×10-5
`potential), 76 V; ring voltage (focusing potential), 320 V;
`
`torr; orifice voltage (declustering
`
`entrance potential, 12 V; collision energy, 25 V; collision
`
`exit potential, 8.0 V. Quantitation was performed by
`
`obtained from the least-square fitted terminal log-linear
`
`portion of the plasma concentration-time profile. The
`
`elimination half-life (T1/2) was calculated as 0.693/kel. The
`area under the curve to the last measurable concentration
`
`(AUC0-t) was calculated by the linear trapezoidal rule. The
`area under the curve extrapolated to infinity (AUCinf) was
`calculated as AUC0-t + (Ct/kel), where Ct is the last
`measurable concentration.8)
`
`Statistical analysis
`For the purpose of bioequivalence analysis AUC0-t and
`Cmax were considered as primary variables. Bioequiva-
`lence was assessed by analysis of variance between
`
`multiple reaction monitoring (MRM) of the protonated
`
`groups (ANOVA) for crossover design and calculating
`
`precursor ion and the related product ion for naltrexone
`
`standard 90% confidence intervals of the ratio test/refer-
`
`using the internal standard method with peak area ratios.
`
`ence. The product were considered bioequivalent if the
`
`The mass transition used for naltrexone and internal
`
`difference between two compared parameters was found
`
`standard were m/z 342 → 324, 328 → 310, respectively
`
`statistically insignificant (P≥0.05) and 90% confidence
`
`(dwell time 150 ms). Quadrupoles Q1 and Q3 were set
`
`intervals for these parameters fell within 80-125%, and
`
`on unit resolution. The analytical data were processed by
`
`the range of equivalence for the non-parameter analysis
`
`Analyst software (version 1.2).
`
`was set to 20% of the reference mean. ANOVA was per-
`
`Extraction of naltrexone from plasma
`
`The naltrexone concentration in plasma was analyzed
`
`using a reported LC/MS/MS method, with slight modifi-
`cation.7) 1 ml of plasma was extracted with 5 ml of
`methyl tert-butyl ether containing internal standard
`
`formed using logarithmic transformed AUCt and Cmax.
`All statistical comparisons were made using EquivTest
`version 1.0 (Statical Solution Ltd., Sangus, MA, USA).6, 8)
`
`Results and Discussion
`
`(naloxone 250 ng/ml in methanol) for 10 minute. After
`
`HPLC/MS/MS analysis
`
`mixing and centrifugation, the organic phase was trans-
`
`With the LC/MS/MS method, no interference was
`
`ferred and evaporated to dryness under nitrogen stream at
`about 40oC and residue was reconstituted in 100 µl of 0.1
`% fomic acid in acetonitrile. After brief mixing for 1min
`
`observed in human plasma. The retention times for naltr-
`
`exone and the internal standard (naloxone) were approxi-
`
`mately 1.30 min (Fig. 2). The quantification limit for
`
`on a vortex mixer, 5 µl of the reaction mixture was
`
`naltrexone in human plasma was 2 ng/ml, based on a sin-
`
`injected onto the chromatographic column.
`
`gle-to-noise ratio of 5.0. The intra- and inter-day coeffi-
`
`Pharmacokinetic analysis
`
`cients of variation were less than 11.520% and 9.762%,
`
`respectively, for the concentration range from 2 to 50 ng/
`
`The pharmacokinetic analysis was performed using non-
`
`ml.
`
`compartmental methods and
`
`the non-compartmental
`
`parameters were derived using standard method. The
`
`Clinical observation
`
`maximum naltrexone concentration (Cmax) was determined
`by the inspection of the individual drug plasma concentra-
`
`The tolerability of naltrexone 50 mg medication was
`
`acceptable. Clinically relevant or drug-related adverse
`
`tion-time profile. The elimination rate constant (Kel) was
`
`effects were not observed in any of the 22 volunteers.
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`72
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`Kor. J. Clin. Pharm., Vol. 16, No. 1, 2006
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`Fig. 2. Choromatogram of naltrexone. Upper chromatogram is double blank plasma uncontained naltrexone and internal
`standard, middle chromatogram is zero blank plasma contained only naltrexone. Lower chromatogram is plasma sample of
`subject 1 after 1 hour at a single naltrexone 50-mg oral dose.
`
`Pharmacokinetic characteristics
`
`The mean concentration-time profiles for the two
`
`brands of naltrexone 50 mg tablets are shown in Fig. 3
`
`and the pharmacokinetic parameters for both formula-
`
`tions are shown in Table 1. All calculated pharmacoki-
`
`netic parameter values were in good agreement with the
`
`previously reported values. The mean terminal half-life
`
`of naltrexone of reference and test brands was 7.99±5.64
`
`hr and 9.40±5.25 hr, respectively (mean terminal half-life
`
`of two products 8.70±5.43).
`
`Table 1. Pharmacokinetic parameters of naltrexone for two
`brands (mean±S.D., n=22)
`
`Pharmacokinetic
`parameter
`AUCt (ng·hr/ml)
`AUC (ng·hr/ml)
`Cmax (ng/ml)
`Tmax (hr)
`kel (hr-1)
`Cltotal/F(L/hr)
`
`Levia 50 mg
`(Reference)
`43.31±10.72
`67.68±15.56
`12.27±0.57
`1.05±0.47
`0.12±0.04
`768.10±137.68
`
`Traxone 50 mg
`(Test)
`43.45±9.91
`70.15±22.26
`12.01±3.92
`0.90±0.50
`0.10±0.05
`817.16±370.17
`
`Standard bioequivalence analysis
`
`design was properly performed. Significant F-test values
`
`No significant sequence effect was found for any of the
`
`were found between subjects and the subjects’ nested
`
`bioavailability parameters, indicating that the cross-over
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`AMN1039
`IPR of Patent No. 7,919,499
`
`

`

`Evaluation of the Bioequivalence of Two Brands of Naltrexone 50 mg Tablet in Healthy Volunteers
`
`73
`
`for AUCt ans Cmax were 1.008 and 1.005 (test/reference),
`
`respectively, and the parameteric 90% confidence inter-
`
`vals for AUCt and Cmax were 0.8862-1.1398
`
`Conclusion
`
`The Statistical comparision of AUCt and Cmax clearly
`
`indicated no significant difference in the two brands of
`
`naltrexone 50 mg tablet. 90% confidence intervals for the
`
`mean ratio (T/R) of AUCt and Cmax were entirely within
`
`the Korea Food and Drug Administration acceptance
`
`range. Based on the pharmacokinetic and statistical
`
`results of this study, we can conclude that Traxone 50 mg
`
`tablets (Myung In Pharm. Co., Ltd., Seoul, Korea) is
`
`bioequivalent to Levia 50 mg tablets (Jeil Pharm. Co.,
`
`Ltd., Seoul, Korea), and that two products can be consid-
`
`Fig. 3. Time course of the mean plasma concentration in
`healthy subjects after a single naltrexone 50-mg oral dose.
`Each point represents the mean + standard deviation. (n=
`®
` (naltrexone 50 mg reference tablet), ○; Traxone
`22, ●; Levia
`(naltrexone 50 mg test tablet))
`
`sequence (SEQ) for AUCt and Cmax, indicating substan-
`
`ered interchangeable in medical practice.
`
`tial inter-subject variation in the pharmacokinetics of nal-
`
`trexone from the two formulation (table 2). No significant
`
`Acknowledgment
`
`period effect in AUCt or Cmax was detected in this study.
`
`The detailed statistical and bioequivalence analyses of
`
`This study was supported by the contract, Bioequiva-
`
`naltrexone for AUCt and Cmax under the assumptions of
`
`lence assessment of naltrexone 50mg tablets after admin-
`
`multiplicative model are given in table 3. The geometric
`
`istering a single oral dose to healthy volunteers, from
`
`means of the parameters are given for the test and refer-
`
`Myung-In Pharm Co., Ltd., Seoul, Korea.
`
`ence formulations of naltrexone, separately and as com-
`
`bined estimates. The parametric point estimates of the
`
`ratio of geometric mean of test and reference products
`
`
`
`Table 2. Analysis of varience test (α=0.05) for AUCt (log-trans-
`formed) and Cmax (log-transformed) for the sumatriptan
`tablets
`
`ANOVA
`
`Group or Sequence
`Subjects/Group
`Period
`Drug
`
`Log-transformed
`AUCt (F-value)
`0.049 (4.351)
`9.171 (2.124)
`0.470 (4.351)
`0.060 (4.351)
`
`Log-transformed
`Cmax (F-value)
`0.291 (4.351)
`3.515 (2.124)
`1.124 (4.351)
`0.005 (4.351)
`
`Table 3. The 90% confidence intervals and results of
`geometric means on the target pharmacokinetic parameters
`of naltrexone
`
`Geometric means
`Test (T) Reference (R)
`11.450
`11.393
`42.382
`42.040
`
`T/R
`1.005
`1.008
`
`Cmax
`AUCt
`
`90% C.I.
`
`0.886-1.140
`0.952-1.068
`
`References
`
`1. D. Hauschke, V.W. Steinijans, E. Diletti. A distribution-
`free procedure for the statistical analysis of bioequivalence
`studies, int. J. Clin. Pharmacol. Ther. Toxicol. 1990; 28:
`72-78.
`2. H.U. Schulz, V.W. Steinijans. Striving for standards in bio-
`equivalence assessment: a review, Int. J. Clin. Pharmacol.
`Ther. Toxicol. 1992; 30: S1-S6.
`3. M. Heiling, M. Egli. Pharmacological treatment of alcohol
`dependence: Target symptoms and target mechanisms.
`Phamacol. Thetapeut., in press.
`4. Gantt Galloway, Monika Koch, Ryan Cello, et al.
`Pharmacokinetics, safety, and tolerability of a depot
`formulation of naltrexone in alcoholics: an open-label
`trial. BMC Psychiatry, 2005; 5: (online) http://www.
`biomedcentral.com/1471-244X/5/18.
`5. St. Louis. MO 63134 U.S.A. Mallinckrodt. Inc. (online)
`www.pharmaceuticals.mallinckrodt.com/_attachments/
`PackageInserts/06-Depade%2025_50_100.pdf
`
`AMN1039
`IPR of Patent No. 7,919,499
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`

`

`74
`
`Kor. J. Clin. Pharm., Vol. 16, No. 1, 2006
`
`6. H.Y. Yun, I.H. Baek, K.I. Kwon. Evaluation of the bio-
`equivalence of sumatriptan in healthy volunteers. Kor. J.
`Clin. Pharm. 2005; 15(2): 160-164.
`7. W. Naidong, Haizhi Bu, Y.L. Chen, et al. Stimultaneous
`development of six LC-MS-MS methods
`for
`the
`determination of multiple analytes in human plasma. J.
`
`Pharmaceut. Biomed. 2002: 28: 1115-1126.
`8. H.A. Dugger, J.D. Carlson, W. Henderson, et al. Bio-
`equivalence evaluation of lansoprazole 50 mg capsules
`(Lanfast® and Lanzor®) in healthy volunteers. Eur. J.
`Pharm. Biopharm. 2001; 51: 153-157.
`
`AMN1039
`IPR of Patent No. 7,919,499
`
`