- Email: [email protected]
Stable-Isotope Methodology in the Bioavailability Study of 17α-Methyltestosterone using Gas Chromatography-Mass Spectrometry
Stable-Isotope Methodology in the Bioavailability Study of 17wMethyltestosterone using Gas Chromatography-Mass Spectrometry YOSHIHIKO SHINOHARA*~, SHIGEO BABA*,YASUJIKASUYA*, VINOD P. SHAH*,AND IRWIN L. HONIGBERG§
GENEKNAPP*, FRANCIS R.
PELSOR*,
Received April 17, 1985, from the 'Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, To o 192-03,Japan, the *Division of Biopharmaceotics, Food and Dru Administration, Rockville, MD 20857,and the §Department of edicinal Chemistry, College of Pharmacy, Unrversity of Georgia, Athens, G i 30602. Accepted for publication November 15. 1985.
x;
other chemicals and solvents were analytical grade and were used without further purification. A p p a r a t u g G a s chromatography-mass spectrometry with selectone is described. Eight healthy male subjects were administered orally a ed ion monitoring (GC-MS-SIM) were performed with on a Shisingle 10-mg 17a-methyltestosteronetablet together with a 10-mg 17amethyltestosterone-4 solution. The serum concentrations of 17a-meth- madzu LKB-9000B gas chromatograph-mass spectrometer equipped with a Shimadzu high speed multiple-ion detector-peak matcher yltestosterone and 17a-methyltestosterone-~were determined by gas 9060s (Shimadzu Seisakusho LM., Kyoto). The conditions of the GCchromatography-mass spectrometry with selected ion monitoring using 17amethyltestosterone-$ as an internal standard. The extent of ab- MS-SIM were the same as those described in a previous paper.16 The electron energy was set at 20 eV. The multiple-ion detector was sorption from the tablet formulation was comparable to that from the oral focused on the ions at d z 302. 305, and 308. The GC had a glass solution. The stable-isotope methodology was compared with the concolumn (1 m x 3 mm i.d.) packed with 1.5% SE-30. The column ventional cross-over method for evaluating the bioavailability of 17atemperature was 230°C, and helium was used as a carrier gas at a methyltestosterone. flow rate of 25 mllmin. Helium was removed using a jet separator. Drug Administration-Each of the eight healthy male volun17a-Methyltestosterone (17P-hydroxy-17a-methyl-4-an- teers,2 1 3 4 years of age, fell within 10% of his ideal body weight for his age and height. No subject had any history of or present drosten-3-one) is a synthetic androgen which has been used cardiovascular, hepatic, GI, testicular, or hemopoietic disease. In in the treatment of eunuchism, eunuchoidism, male impoaddition, the subjects had to refrain from all drugs for 7 d prior to tence, and female breast cancer. It is an orally effective each study day and 3 d following drug administration. Informed hormone analogue that has considerably higher oral activity consent was obtained from all subjects. than testosterone.14 The study was carried out in a two-way cross-over fashion. The drug was administered on two separate occasions a week apart. After The bioavailabilityhioequivalency regulations of the Unitan 8-h overnight fast, each subject was administered orally either a ed States Food and Drug Administration (FDA), which 10-mg 17a-methyltestosterone tablet with a reference solution (100 became effective on July 7, 1977, listed 110 drugs and drug mL) containing 10 mg of 17u-methyltestosterone-d3or a solution dosage forms which were known or suspected of having (100 mL) containing 10 mg of 17a-methyltestosterone with a referpotential bioavailabilityhioequivalency problems.6 17aence solution (100 mL) containing 10 mg of 17a-methyltestosteroneMethyltestosterone is one of the drugs listed because differd3. The tablet was administered with 120 mL of water, and the ences in product bioavailability between different routes of solution was followed by a 20-mL water rinse of the container. No administration, e.g., oral, buccal, and sublingual, have not food was permitted for 4 h following drug administration. Blood (17 been evaluated. Furthermore, there appears to be little mL) was drawn just before the oral dose and at various times postdose (Table I). After clotting, the samples were centrifuged to information on its pharmacokinetic characteristics. Alkalay separate the serum fraction. The serum samples were stored at and co-workers6~7used a spectrophotofluorometric determi-20°C until the time of assay. nation to assess the relative bioavailability of l7a-methyltesAnalysis of Serum Samples-The procedure used for the analysis tosterone formulations in a five-way cross-over design. of serum samples has been described elsewhere.18 Briefly, to each The use of gas chromatography-mass spectrometry (GC1.0-mL serum sample, 50 ng of 17a-methylteStoSterone-d6was added MS) and stable-isotope-labeled drugs as diluents has found a as an analytical internal standard, and the sample was extracted broad application in pharmacological studies.s1l Recently, with n-hexane. After the solvent was evaporated, the dried residue studies on the relative bioavailability of several different was dissolved in 20 FL of n-hexane. A 2-4-pL aliquot of the solution formulations of the same drug have been performed effectivewas subject to GC-MS-SIM. The concentrations of 17a-methyltestosterone and 17a-methyltestosterone-d3in serum were calculated ly by use of a stable-isotope-labeled analogue as the reference with which the test, unlabeled formulations are ~ 0 r n p a r e d . l ~ ~by~ determining the peak height ratios of mlz 302 verus m/z 308 (ddd,) and mlz 305 versus m/z 308 (ddd6). The relative bioavailability of 17a-methyltestosterone tabData AnalysibPeak serum concentration) ,C( and the time to let formulations with coadministration of a stable-isotopeachieve maximum serum concentration (t-1 were determined dilabeled 17a-methyltestosterone solution as an internal biorectly from the observed data. The estimate of the elimination halflogical standard is described in this paper. life (tllz)was obtained from at least four points of the terminal loglinear portion of the curve. The area under the serum concentration time curve (AUC) was calculated by the trapezoidal method. The Experimental Section area under the first moment of the curve (AUMC) was computed M a t e r i a l s T h e syntheses of 17a-methyltestosterone-d3(17ghysimilarly by multiplying each individual serum concentration by its droxy-17a-methyl-d3-4-androsten-3-one) and 17a-methyltestostertime. Extrapolations through infinity were calculated from the one-d6 (17~hydroxy-17a-methyl-d3-4-androsten-19-d3-3-one) were following equation using the last detectable serum concentration at previously reported.15 The isotopic compositions were 99.6% atoms of time t (Ct;8-h point) and the terminal elimination rate constant (A = deuterium (4: 98.64%, d2: 1.36%, dl:0.00%) for 17a-methyltestosterone-d3, and 99.3% atoms of deuterium (4:96.88%, d6:2.268, d4: 0.86%) for 17a-methyltestosterone-d6. Reagent-grade unlabeled 17amethyTtestosterone (Tokyo Kasei Kogyo, Tokyo) was recrystallized from n-hexane:ethyl acetate (5:l). 17a-Methyltestosterone tablets (10 mg, Ciba Geigy, lot no. 10190) were commercial products. All Abatracl El The application of a stable-isotope coadministration technique for estimating the relative bioavailability of 17a-methyltestoster-
W22-3549/86/02OO-O 16 1$0l.OO/O 0 1986,American Pharmaceutical Association
Journal of Pharmaceutical Sciences / 161 Vol. 75, No. 2, February 1986
Table CSbrum Concentratlonr of 17~Methyltertoatetrone(4Solutlon Versus A Solution)
Time, h 0.33 0.67 1.o 1.5 2.0 2.5 3.0 4.0 6.0 8.0 t14, h AUCo-, pmol * h/mL
Time, h 0.33 0.67 1.o 1.5 2.0 2.5 3.0 4.0 6.0 8.0 tlfiv h AUCc-,
Subject 1, ng/mL
Subject 3, nglrnL
Subject 2, ng/mL
Subject 4, ng/mL
4
4
Ratio
dl
4
Ratio
4
4
Ratio
do
4
Ratio
24.8 51.6 30.7 12.7 8.55 6.38 5.27 3.88 2.41 0.77
22.7 48.1 27.6 11.1 7.96 5.89 4.80 3.74 2.15 0.72
1.09 1.07 1.11 1.14 1.07 1.08 1.10 1-04 1.12 1.07
43.7 24.3 13.9 8.26 5.58 4.06 3.07 2.23 0.95 0.48
41.9 23.4 13.8 7.84 5.41 3.75 2.83 2.06 0.89 0.48
1.04 1.04 1.01 1.05 1.03 1.08 1.08 1.08 1.07 1.00
42.8 36.4 18.9 11.4 7.02 5.33 4.81 3.40 1.28 0.43
42.2 35.9 18.4 11.4 6.82 4.94 4.05 3.37 1.72 0.39
1.01 1.01 1.03 1.00 1.03 1.08 1.19 1.01 0.74 1.11
33.5 18.4 11.0 6.16 3.80 2.72 1.63 1.31 0.57 0.28
32.1 17.7 10.5 5.82 3.76 2.55 1.60 1.23 0.65 0.22
1.04 1.04 1.05 1.06 1.01 1.07 1.02 1.07 0.88 1.27
1.91 231.4
1.90 210.0
1.01 1.09
1.08 154.4
1.86 146.6
0.97 1.04
1.49 194.8
1.58 191.3
0.94 1.01
1.91 111.4
1.73 105.1
1.10 1.05
Subject 5, ng/mL
Subject 6, ng/mL
Subject 7, ng/mL
Subject 8, ng/mL
do
4
Ratio
do
4
Ratio
4
4
Ratio
do
4
62.0 26.0 16.7 10.4 6.16 4.00 3.15 2.26 1.41 0.97
56.3 24.3 15.3 10.4 5.86 3.52 2.96 2.12 1.19 0.91
1.10 1.07 1.09 1.oo 1.05 1.14 1.06 1.07 1.18 1.07
42.0 36.2 23.9 13.9 8.83 6.49 5.65 3.88 1.80 0.95
41.9 30.0 22.9 13.3 8.76 6.11 5.05 3.45 1.73 0.89
1.oo 1.01 1.04 1.05 1.01 1.06 1.12 1.12 1.04 1.07
-
60.1 37.9 27.3 11.6 7.90 6.70 4.87 3.71 2.32 1.07
59.1 37.5 26.2 11.3 7.46 6.05 4.39 3.46 1.91 1.05
1.02 1.01 1.04 1.03 1.06 1.11 1.11 1.07 1.21 1.02
17.9 27.5 28.6 25.3 27.3 16.6 12.7 6.86 2.90 1.58
17.4 26.2 26.8 23.4 25.6 15.3 12.2 6.20 2.79 1.47
1.03 1.05 1.07 1.08 1.07 1.06 1.04 1.10 1.04 1.07
2.99 199.2
2.91 181.5
1.03 1.09
1.94 224.6
1.98 214.3
0.98 1.04
2.33 251.4
2.28 236.9
1.02 1.05
1.68 305.5
1.68 283.7
1.00 1.07
Ratio
pmol. h/mL
AUMCo,
= AUMCe,
tC, ct ++A A2
The mean residence time (MRT) was calculated as follows:
MRT =
AUMCo, AUCo,,
(3)
The difference in the MRT between an oral tablet dosage form and an oral solution has been termed the mean in vivo dissolution time (MDT) by Riegelman and Collier."
(4) MDT = MRTtablet - MRTsolution The relative extent of absorption (F,,) was calculated by the following equation:
where D is the administered dose and the subscript refers to the treatment.
Results and Discussion The most commonly applied approach to bioavailability assessment uses a cross-over design. The method is based on the assumption that total body clearance of the individual remains unchanged from one test dose to another administered on a separate occasion. The AUC ratio arising from test and reference doses is a measure of bioavailability. If there is evidence that the clearance but not the volume of distribution varies in the individual, the AUC multiplied by the terminal distribution rate constant (A) can be used to gain a 162 / Journal of Pharmaceutical Sciences Vol. 75, No. 2, February 7986
more precise index of bioavailability than obtainable from the AUC alone. Because of intrasubject variability in drug disposition, however, it is usually the case that a large number of subjects are required to assess bioavailability. The use of the stable-isotope methodology in bioavailability studies involves the simultaneous administration, to the same individual, of the formulated drugs and an oral (or intravenous) solution of a stable-isotope-labeled analogue of the drug. This procedure offers significant advantages over traditiohal techniques since the method accounts for intraindividual differences in absorption, distribution, and elimination. It was essential to establish the bioequivalence of 17amethyltestosterone-d3 and 17a-methyltestosterone before applying the present stable-isotope methodology for the bioavailability study. A procedure used to verify the absence of an in vivo isotope effect with 17a-methyltestosterone-d3 involved oral administration of a 10-mg plus 10-mg mixture of 17a-methyltestosterone and 17a-methyltestosterone-d3solution to eight healthy subjects. Analysis of serum samples for both 17a-methyltestosterone and 17a-methyltestosterone-d3 allowed us to study the fate of 17a-methyltestosterone-d3 independently from 17a-methyltestosterone given on a single-dose schedule. The results presented in Table I show that the observed ratios of the two forms (17a-methyltestosterone/l7a-methyltestosterone-d3)in serum samples were virtually identical with the expected ratio throughout the sample-collectionperiod. Also, the half-life and AUC of 17amethyltestosterone-d3 were practically the same as those of 17a-methyltestosterone. From these results, the pharmacokinetics of 17a-methyltestosterone-d3could be considered to be equivalent to that of the unlabeled methyltestosterone. Methods used to evaluate the absorption kinetics of a drug after oral administration remain controversia1.'8 However, a
quantitative assessment of absorption data is usually made by comparing the times (tmax)required to reach the peak concentration (Cmm)of drug following oral administration. Figure 1 shows the individual serum concentration-time curves of 17a-methyltestosterone and l7a-methyltestosterone-& after oral dosing with a 10-mg 17a-methyltestosterone tablet together with 10 mg of a 17a-methyltestosteroned3solution. The calculated pharmacokinetic parameters are presented in Table 11.17a-Methyltestosteroneand l7a-methyltestosterone-d3 were absorbed rapidly. The t,, values ranged from 0.67 to 1.5 h for the tablet formulation and from 0.33 to 0.67 h for the solution. The C,, values for the 17amethyltestosterone tablet and the 17a-methyltestosteroned3 solution preparations ranged from 11.4 to 63.6 and 20.6 to 67.8 ng/mL, respectively. The mean terminal half-life values in eight subjects were 2.29 f 0.51 h for the tablet and 2.38 f 0.32 h for the solution, which were not significantly different. These tmu, C, and half-life values were similar to those reported by other workers who measured l7a-methyltestosterone serum concentrations by the spectrophotofluorometric method.' However, it is often difficult to define precisely the peak time because only a limited number of blood samples were in general obtainable from a human study. The statistical moment method is one of the preferable alternatives because it is easily computed and has potentially smaller error. The statistical moment method has recently been developed in pharmacokinetics as a concept to estimate the time involved in the absorption processes.l'Jg In an application of the concept, it has been shown that the mean in vivo dissolution time (MDT)is the most useful index of evaluating absorption In the present stable-isotope methodology, both tablet and solution dosage forms were administered simultaneously to the same subjects. It is then reasonable to assume that once 17a-methyltestosterone is released from the tablet dosage form, it behaves in the same manner as 17a-methyltestosterone-d3 administered in solution. Therefore, the difference in 100,
Table ICPharmacoklnetic Parameters 17a-Methyltestosterone-dj Solution
17a-Methyltestosterone
Tablet
Subject
1.o
Mean 2
SD
tmaxs
Crnax,
h
ng/mL
'+m
1.5 0.67 1.o 1.5 1.o 1.o 0.67
23.6 20.2 17.7 11.4 20.0 63.6 17.0 15.4
2.28 1.86 2.72 2.38 1.49 2.01 2.48 3.13
0.33 0.67 0.33 0.33 0.33 0.33 0.33 0.67
35.2 54.8 27.8 20.6 58.6 67.8 34.4 36.8
2.85 2.31 2.55 2.31 1.74 2.30 2.51 2.46
1.04 0.32
23.6 16.6
2.29 0.51
0.42 0.16
42.0 16.5
2.38 0.32
their mean residence times (MRT) may adequately provide the MDT from the tablet dosage form. The MRT and MDT calculated from eqs. 3 and 4 are shown in Table 111. The MRT of 17a-methyltestosterone tablet and 17a-methyltestosterone-d3 solution indicated a statistically significant increase in the MRT for the tablet in each subject. The MDT, which probably reflects the dissolution rate or the release rate of 17a-methyltestosterone from the tablet in the GI tract, varied widely among the subjects, ranging from 0.12 to 2.34 h. It is likely that the dissolution of 17a-methyltestosterone from the tablet form is affected by interindividual differences in GI motility to a large extent. The MDT data based on the conventional cross-over method are presented in Table IV for comparison. The relative bioavailability data by the conventional method were calculated using the 17a-methyltestosterone tablet data in the stable-isotope coadministration study and the 17a-methyltestosterone solution data in the isotope-effect study on different occasions separated by 1 week. With intra-individual variability in the disposition of
1 Subiect 3
Subject 4
.:
100.
<.
50
(II
Subject 8
i
.-o
10
m
. = 5 U al
0
s
O
1
& 0.5 v)
0
7
8
0
1
-2
3
4
6
Time, h
8
0
1
2
3
4
8
8
0
1
2
3
4
6
8
Time, h
Figure 1-Serum concentration-time cufves for 7 7a-met~yltestosterone(+) and 7 7a-methylte~tosterone-d~ (---H---)after a single oral dose of a 70-mg 7 7a-methyltestosterone tablet and a 70-mg 7 7 ~ - m e t h y l r e s r o s t e - d solution. ~ Journal of Pharmaceutical Sciences / 163 Vol. 75, No. 2, February 7986
Table IlCRelatlve Bloavallablllty of 17rrMethylteatoaterone Tablet by the Stable-Isotope Coadmlnlstratlon Technique
Mean 2
SD
MRT, h
F,,,
Subject
YO
Tablet
Solution
193.9 176.2 134.1 109.6 184.7 401.7 174.6 209.4
192.2 275.0 135.9 97.1 201.8 353.3 139.7 192.3
198.0 88.4
198.4 82.4
Table V-lntra-lndlvldual Varlatlona In the Pharmacoklnetlcs of 17a-Methyltestosterone4E,Solutlon
MDT, h
Tablet
Solution
99.9 63.5 97.8 111.8 90.6 112.5 123.7 107.8
3.36 3.21 2.98 3.59 2.88 2.58 3.32 4.70
2.51 2.07 2.55 2.17 1.56 2.47 2.20 2.36
1.42 1.31 0.12 1.16 2.34
100.9 18.3
3.32 0.64
2.24 0.32
1.09 0.67
Mean f SD
0.85 1.13
0.43
F~I,
MDT, h
83.8 114.1 68.9 101.6 92.7 178.8 69.4 68.5
1.21 1.47 1.23 2.01 0.66 0.47 1.15 2.09
97.2 37.0
1.29 0.57
17a-methyltestosterone, the MDT estimated from the conventional method may be less meaningful. Using the serum concentration-time data obtained from the present stable-isotope coadministration method, the mean AUC was calculated to be 198.0 f 88.4 pmol/ml/h for the 10-mg 17a-methyltestosterone tablet and 198.4 82.4 pmol/mL/h for the 10-mg 17a-methyltestosterone-d3solution (Table 111).The relative bioavailability values (Fre,)ranged from 90.6 to 123.7%in seven of the eight subjects tested. One subject gave a considerably low Frel value of 63.5%, for unknown reasons. Except for this subject, the tablet formulation tested was demonstrated to be equivalent to the oral solution with respect to the extent of absorption. That is, the rate of dissolution did not affect the extent of absorption. We then compared the relative bioavailability obtained by the present stable-isotope coadministration technique with that obtained by the conventional cross-over method (Tables I11 and IV). The mean relative bioavailability among the eight subjects was 97.2% for the conventional method and 100.9% for the stable-isotope coadministration method; the values were practically identical. The CV for the conventional method (38.0%)was -20% higher than that for the stableisotope coadministration method (18.1%), which confirmed the reliability of the stable-isotope coadministration method. The reason for this difference can apparently be attributed to the fundamental assumption of constant clearance in the cross-over method. Significant intra-individual variability in the pharmacokinetic behavior of 17a-methyltestosterone was observed between the two methods of administering 17amethyltestosterone-d3 solution 1 week apart (Table V). For
*
164 / Journal of Pharmaceutical Sciences Vol. 75, No. 2, February 1986
MRT
AUC~~~
Table IV-Relatlve Bloavallablllty of 17a-Methyltestosterone Tablet by the Cross-Over Method
Subject
Week 1Mleek 2
Subject 1 2 3 4 5 6 7 8
~
1.09 0.53 1.41 1.08 1.11 1.65 0.59 0.68
0.86
0.84 0.72 0.70 0.72 1.19 1.06 0.91
example, changes in clearances were indicated by a large deviation in the ratio of the AUC of 17a-methyltestosteroned3 solution in the first week to that in the second week. Therefore, in assessing bioavailability, the assumption of constant clearance in the individual between the occasions of receiving the reference and test dose is suspect for 17amethyltestosterone. The number of subjects required to accurately detect a difference of 20% (a = 0.05, 1 - /3 = 0.8) in the relative bioavailability of two formulations of 17a-methyltestosterone tablets was estimated from the present data according to the method of Heck et al.l2 Although the estimated number of subjects required for a conventional cross-over method was 40,12 subjects were required for the stable-isotope coadministration method.
References and Notes 1. Ruzicka, von L.; Goldgerg, M. W.; Rmenberg, H. R. Helv.Chim. Acta. 1935,18, 1487. 2. Emmens, C. W.; Parkes, A. S. J . End~crinol. 1939,1,323. G. L. Br. Med. J . 1939,2, 11. 3. FOES, 4. Lisser. H.: Curtis. L. E. J . Clin. Endocrinol. 1943.3. 389. 5. Fed. Regist. 1977; 42, 1624. 6. Alkalay, D.; Khemani, L.; Bartlett, M. F. J . Pharm. Sci. 1972, 61. --,1746. -. 7. Alkalay, D.; Khemani, L.; Wagner, W. E.; Bartlett, M. F. J . Clin. Phurm. 1973,13, 142. 8. Baillie, T. A. Phurmacol. Rev. 1981,33, 81. 9. Garland, W. A.; Powell, M. L. J . Chromatogr. Sci. 1981,19,392. 10. Eichelbaum, M.; von Unruh, G. E.;Somogyi, A. Clin.Pharmacokinet. 1982, 7, 490. 11. Murphy, P. J.; Sullivan, H. R. Ann. Rev. P h u r m o l . Taricol. 1980,20, 609. 12. Heck, H. A.; Buttrill, S. E.; Flynn, N. W.; D er, R. L.; Anbar, M.; I
,
Cairns, T.; Dighe, S.; Cabana, B. E. J . $hurmokinet. Biophurm. 1979, 7, 233. 13. Alkalay, D.; Wagner, W. E.; Carlsen, S.; Khemani, L.; Volk, J.; Bartlett, M. F.; LeSher, A. Clin.Pharmacol. Ther. 1980,27,697. 14. Eichelbaum, M.; Dengler, H. J.; Somogyi, A.; von Unruh, G. E. Eur. J . Phurmacol. 1981.19. 127. 15. Shinohara, Y.; Baba, S.yKaauya, Y. Steroids 1984,44, 253. 16. Shinohara, Y.; Baba, S.; Kasuya, Y. J . Chromatogr. 1985,338,
281. 17. Riegelman, S.; Collier, P. J . Phurmacokinet. Biopharm. 1980,8, 509. 18. Gibaldi, M. “Bio harmaceutics and Clinical Pharmacokinetics”; Lea & Febi er: fhiladelphia, 1984; p 135-137. 19. Yamaoka, Nakagawa, T.; Uno, J . Phurmacokinet. Biohurm. 1978,6,547. 20. &anigawara,Y.; Yamaoka, K.; Nakagawa, T.; Uno, T. J . Pharm. Sci. 1982, 71, 1129. 21. Gillespie, W. R.; DiSanto, A. R.; Monovich, R. E.; Albert, K. S . J . Phurm. Sci. 1982, 71,1034. 22. Graffner, C.; Nicklasson, M.; Lindgren, J. J . Phurmacokinet. Biophurm. 1984,12, 367.
k;
f.
GENEKNAPP*, FRANCIS R.
PELSOR*,
Received April 17, 1985, from the 'Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, To o 192-03,Japan, the *Division of Biopharmaceotics, Food and Dru Administration, Rockville, MD 20857,and the §Department of edicinal Chemistry, College of Pharmacy, Unrversity of Georgia, Athens, G i 30602. Accepted for publication November 15. 1985.
x;
other chemicals and solvents were analytical grade and were used without further purification. A p p a r a t u g G a s chromatography-mass spectrometry with selectone is described. Eight healthy male subjects were administered orally a ed ion monitoring (GC-MS-SIM) were performed with on a Shisingle 10-mg 17a-methyltestosteronetablet together with a 10-mg 17amethyltestosterone-4 solution. The serum concentrations of 17a-meth- madzu LKB-9000B gas chromatograph-mass spectrometer equipped with a Shimadzu high speed multiple-ion detector-peak matcher yltestosterone and 17a-methyltestosterone-~were determined by gas 9060s (Shimadzu Seisakusho LM., Kyoto). The conditions of the GCchromatography-mass spectrometry with selected ion monitoring using 17amethyltestosterone-$ as an internal standard. The extent of ab- MS-SIM were the same as those described in a previous paper.16 The electron energy was set at 20 eV. The multiple-ion detector was sorption from the tablet formulation was comparable to that from the oral focused on the ions at d z 302. 305, and 308. The GC had a glass solution. The stable-isotope methodology was compared with the concolumn (1 m x 3 mm i.d.) packed with 1.5% SE-30. The column ventional cross-over method for evaluating the bioavailability of 17atemperature was 230°C, and helium was used as a carrier gas at a methyltestosterone. flow rate of 25 mllmin. Helium was removed using a jet separator. Drug Administration-Each of the eight healthy male volun17a-Methyltestosterone (17P-hydroxy-17a-methyl-4-an- teers,2 1 3 4 years of age, fell within 10% of his ideal body weight for his age and height. No subject had any history of or present drosten-3-one) is a synthetic androgen which has been used cardiovascular, hepatic, GI, testicular, or hemopoietic disease. In in the treatment of eunuchism, eunuchoidism, male impoaddition, the subjects had to refrain from all drugs for 7 d prior to tence, and female breast cancer. It is an orally effective each study day and 3 d following drug administration. Informed hormone analogue that has considerably higher oral activity consent was obtained from all subjects. than testosterone.14 The study was carried out in a two-way cross-over fashion. The drug was administered on two separate occasions a week apart. After The bioavailabilityhioequivalency regulations of the Unitan 8-h overnight fast, each subject was administered orally either a ed States Food and Drug Administration (FDA), which 10-mg 17a-methyltestosterone tablet with a reference solution (100 became effective on July 7, 1977, listed 110 drugs and drug mL) containing 10 mg of 17u-methyltestosterone-d3or a solution dosage forms which were known or suspected of having (100 mL) containing 10 mg of 17a-methyltestosterone with a referpotential bioavailabilityhioequivalency problems.6 17aence solution (100 mL) containing 10 mg of 17a-methyltestosteroneMethyltestosterone is one of the drugs listed because differd3. The tablet was administered with 120 mL of water, and the ences in product bioavailability between different routes of solution was followed by a 20-mL water rinse of the container. No administration, e.g., oral, buccal, and sublingual, have not food was permitted for 4 h following drug administration. Blood (17 been evaluated. Furthermore, there appears to be little mL) was drawn just before the oral dose and at various times postdose (Table I). After clotting, the samples were centrifuged to information on its pharmacokinetic characteristics. Alkalay separate the serum fraction. The serum samples were stored at and co-workers6~7used a spectrophotofluorometric determi-20°C until the time of assay. nation to assess the relative bioavailability of l7a-methyltesAnalysis of Serum Samples-The procedure used for the analysis tosterone formulations in a five-way cross-over design. of serum samples has been described elsewhere.18 Briefly, to each The use of gas chromatography-mass spectrometry (GC1.0-mL serum sample, 50 ng of 17a-methylteStoSterone-d6was added MS) and stable-isotope-labeled drugs as diluents has found a as an analytical internal standard, and the sample was extracted broad application in pharmacological studies.s1l Recently, with n-hexane. After the solvent was evaporated, the dried residue studies on the relative bioavailability of several different was dissolved in 20 FL of n-hexane. A 2-4-pL aliquot of the solution formulations of the same drug have been performed effectivewas subject to GC-MS-SIM. The concentrations of 17a-methyltestosterone and 17a-methyltestosterone-d3in serum were calculated ly by use of a stable-isotope-labeled analogue as the reference with which the test, unlabeled formulations are ~ 0 r n p a r e d . l ~ ~by~ determining the peak height ratios of mlz 302 verus m/z 308 (ddd,) and mlz 305 versus m/z 308 (ddd6). The relative bioavailability of 17a-methyltestosterone tabData AnalysibPeak serum concentration) ,C( and the time to let formulations with coadministration of a stable-isotopeachieve maximum serum concentration (t-1 were determined dilabeled 17a-methyltestosterone solution as an internal biorectly from the observed data. The estimate of the elimination halflogical standard is described in this paper. life (tllz)was obtained from at least four points of the terminal loglinear portion of the curve. The area under the serum concentration time curve (AUC) was calculated by the trapezoidal method. The Experimental Section area under the first moment of the curve (AUMC) was computed M a t e r i a l s T h e syntheses of 17a-methyltestosterone-d3(17ghysimilarly by multiplying each individual serum concentration by its droxy-17a-methyl-d3-4-androsten-3-one) and 17a-methyltestostertime. Extrapolations through infinity were calculated from the one-d6 (17~hydroxy-17a-methyl-d3-4-androsten-19-d3-3-one) were following equation using the last detectable serum concentration at previously reported.15 The isotopic compositions were 99.6% atoms of time t (Ct;8-h point) and the terminal elimination rate constant (A = deuterium (4: 98.64%, d2: 1.36%, dl:0.00%) for 17a-methyltestosterone-d3, and 99.3% atoms of deuterium (4:96.88%, d6:2.268, d4: 0.86%) for 17a-methyltestosterone-d6. Reagent-grade unlabeled 17amethyTtestosterone (Tokyo Kasei Kogyo, Tokyo) was recrystallized from n-hexane:ethyl acetate (5:l). 17a-Methyltestosterone tablets (10 mg, Ciba Geigy, lot no. 10190) were commercial products. All Abatracl El The application of a stable-isotope coadministration technique for estimating the relative bioavailability of 17a-methyltestoster-
W22-3549/86/02OO-O 16 1$0l.OO/O 0 1986,American Pharmaceutical Association
Journal of Pharmaceutical Sciences / 161 Vol. 75, No. 2, February 1986
Table CSbrum Concentratlonr of 17~Methyltertoatetrone(4Solutlon Versus A Solution)
Time, h 0.33 0.67 1.o 1.5 2.0 2.5 3.0 4.0 6.0 8.0 t14, h AUCo-, pmol * h/mL
Time, h 0.33 0.67 1.o 1.5 2.0 2.5 3.0 4.0 6.0 8.0 tlfiv h AUCc-,
Subject 1, ng/mL
Subject 3, nglrnL
Subject 2, ng/mL
Subject 4, ng/mL
4
4
Ratio
dl
4
Ratio
4
4
Ratio
do
4
Ratio
24.8 51.6 30.7 12.7 8.55 6.38 5.27 3.88 2.41 0.77
22.7 48.1 27.6 11.1 7.96 5.89 4.80 3.74 2.15 0.72
1.09 1.07 1.11 1.14 1.07 1.08 1.10 1-04 1.12 1.07
43.7 24.3 13.9 8.26 5.58 4.06 3.07 2.23 0.95 0.48
41.9 23.4 13.8 7.84 5.41 3.75 2.83 2.06 0.89 0.48
1.04 1.04 1.01 1.05 1.03 1.08 1.08 1.08 1.07 1.00
42.8 36.4 18.9 11.4 7.02 5.33 4.81 3.40 1.28 0.43
42.2 35.9 18.4 11.4 6.82 4.94 4.05 3.37 1.72 0.39
1.01 1.01 1.03 1.00 1.03 1.08 1.19 1.01 0.74 1.11
33.5 18.4 11.0 6.16 3.80 2.72 1.63 1.31 0.57 0.28
32.1 17.7 10.5 5.82 3.76 2.55 1.60 1.23 0.65 0.22
1.04 1.04 1.05 1.06 1.01 1.07 1.02 1.07 0.88 1.27
1.91 231.4
1.90 210.0
1.01 1.09
1.08 154.4
1.86 146.6
0.97 1.04
1.49 194.8
1.58 191.3
0.94 1.01
1.91 111.4
1.73 105.1
1.10 1.05
Subject 5, ng/mL
Subject 6, ng/mL
Subject 7, ng/mL
Subject 8, ng/mL
do
4
Ratio
do
4
Ratio
4
4
Ratio
do
4
62.0 26.0 16.7 10.4 6.16 4.00 3.15 2.26 1.41 0.97
56.3 24.3 15.3 10.4 5.86 3.52 2.96 2.12 1.19 0.91
1.10 1.07 1.09 1.oo 1.05 1.14 1.06 1.07 1.18 1.07
42.0 36.2 23.9 13.9 8.83 6.49 5.65 3.88 1.80 0.95
41.9 30.0 22.9 13.3 8.76 6.11 5.05 3.45 1.73 0.89
1.oo 1.01 1.04 1.05 1.01 1.06 1.12 1.12 1.04 1.07
-
60.1 37.9 27.3 11.6 7.90 6.70 4.87 3.71 2.32 1.07
59.1 37.5 26.2 11.3 7.46 6.05 4.39 3.46 1.91 1.05
1.02 1.01 1.04 1.03 1.06 1.11 1.11 1.07 1.21 1.02
17.9 27.5 28.6 25.3 27.3 16.6 12.7 6.86 2.90 1.58
17.4 26.2 26.8 23.4 25.6 15.3 12.2 6.20 2.79 1.47
1.03 1.05 1.07 1.08 1.07 1.06 1.04 1.10 1.04 1.07
2.99 199.2
2.91 181.5
1.03 1.09
1.94 224.6
1.98 214.3
0.98 1.04
2.33 251.4
2.28 236.9
1.02 1.05
1.68 305.5
1.68 283.7
1.00 1.07
Ratio
pmol. h/mL
AUMCo,
= AUMCe,
tC, ct ++A A2
The mean residence time (MRT) was calculated as follows:
MRT =
AUMCo, AUCo,,
(3)
The difference in the MRT between an oral tablet dosage form and an oral solution has been termed the mean in vivo dissolution time (MDT) by Riegelman and Collier."
(4) MDT = MRTtablet - MRTsolution The relative extent of absorption (F,,) was calculated by the following equation:
where D is the administered dose and the subscript refers to the treatment.
Results and Discussion The most commonly applied approach to bioavailability assessment uses a cross-over design. The method is based on the assumption that total body clearance of the individual remains unchanged from one test dose to another administered on a separate occasion. The AUC ratio arising from test and reference doses is a measure of bioavailability. If there is evidence that the clearance but not the volume of distribution varies in the individual, the AUC multiplied by the terminal distribution rate constant (A) can be used to gain a 162 / Journal of Pharmaceutical Sciences Vol. 75, No. 2, February 7986
more precise index of bioavailability than obtainable from the AUC alone. Because of intrasubject variability in drug disposition, however, it is usually the case that a large number of subjects are required to assess bioavailability. The use of the stable-isotope methodology in bioavailability studies involves the simultaneous administration, to the same individual, of the formulated drugs and an oral (or intravenous) solution of a stable-isotope-labeled analogue of the drug. This procedure offers significant advantages over traditiohal techniques since the method accounts for intraindividual differences in absorption, distribution, and elimination. It was essential to establish the bioequivalence of 17amethyltestosterone-d3 and 17a-methyltestosterone before applying the present stable-isotope methodology for the bioavailability study. A procedure used to verify the absence of an in vivo isotope effect with 17a-methyltestosterone-d3 involved oral administration of a 10-mg plus 10-mg mixture of 17a-methyltestosterone and 17a-methyltestosterone-d3solution to eight healthy subjects. Analysis of serum samples for both 17a-methyltestosterone and 17a-methyltestosterone-d3 allowed us to study the fate of 17a-methyltestosterone-d3 independently from 17a-methyltestosterone given on a single-dose schedule. The results presented in Table I show that the observed ratios of the two forms (17a-methyltestosterone/l7a-methyltestosterone-d3)in serum samples were virtually identical with the expected ratio throughout the sample-collectionperiod. Also, the half-life and AUC of 17amethyltestosterone-d3 were practically the same as those of 17a-methyltestosterone. From these results, the pharmacokinetics of 17a-methyltestosterone-d3could be considered to be equivalent to that of the unlabeled methyltestosterone. Methods used to evaluate the absorption kinetics of a drug after oral administration remain controversia1.'8 However, a
quantitative assessment of absorption data is usually made by comparing the times (tmax)required to reach the peak concentration (Cmm)of drug following oral administration. Figure 1 shows the individual serum concentration-time curves of 17a-methyltestosterone and l7a-methyltestosterone-& after oral dosing with a 10-mg 17a-methyltestosterone tablet together with 10 mg of a 17a-methyltestosteroned3solution. The calculated pharmacokinetic parameters are presented in Table 11.17a-Methyltestosteroneand l7a-methyltestosterone-d3 were absorbed rapidly. The t,, values ranged from 0.67 to 1.5 h for the tablet formulation and from 0.33 to 0.67 h for the solution. The C,, values for the 17amethyltestosterone tablet and the 17a-methyltestosteroned3 solution preparations ranged from 11.4 to 63.6 and 20.6 to 67.8 ng/mL, respectively. The mean terminal half-life values in eight subjects were 2.29 f 0.51 h for the tablet and 2.38 f 0.32 h for the solution, which were not significantly different. These tmu, C, and half-life values were similar to those reported by other workers who measured l7a-methyltestosterone serum concentrations by the spectrophotofluorometric method.' However, it is often difficult to define precisely the peak time because only a limited number of blood samples were in general obtainable from a human study. The statistical moment method is one of the preferable alternatives because it is easily computed and has potentially smaller error. The statistical moment method has recently been developed in pharmacokinetics as a concept to estimate the time involved in the absorption processes.l'Jg In an application of the concept, it has been shown that the mean in vivo dissolution time (MDT)is the most useful index of evaluating absorption In the present stable-isotope methodology, both tablet and solution dosage forms were administered simultaneously to the same subjects. It is then reasonable to assume that once 17a-methyltestosterone is released from the tablet dosage form, it behaves in the same manner as 17a-methyltestosterone-d3 administered in solution. Therefore, the difference in 100,
Table ICPharmacoklnetic Parameters 17a-Methyltestosterone-dj Solution
17a-Methyltestosterone
Tablet
Subject
1.o
Mean 2
SD
tmaxs
Crnax,
h
ng/mL
'+m
1.5 0.67 1.o 1.5 1.o 1.o 0.67
23.6 20.2 17.7 11.4 20.0 63.6 17.0 15.4
2.28 1.86 2.72 2.38 1.49 2.01 2.48 3.13
0.33 0.67 0.33 0.33 0.33 0.33 0.33 0.67
35.2 54.8 27.8 20.6 58.6 67.8 34.4 36.8
2.85 2.31 2.55 2.31 1.74 2.30 2.51 2.46
1.04 0.32
23.6 16.6
2.29 0.51
0.42 0.16
42.0 16.5
2.38 0.32
their mean residence times (MRT) may adequately provide the MDT from the tablet dosage form. The MRT and MDT calculated from eqs. 3 and 4 are shown in Table 111. The MRT of 17a-methyltestosterone tablet and 17a-methyltestosterone-d3 solution indicated a statistically significant increase in the MRT for the tablet in each subject. The MDT, which probably reflects the dissolution rate or the release rate of 17a-methyltestosterone from the tablet in the GI tract, varied widely among the subjects, ranging from 0.12 to 2.34 h. It is likely that the dissolution of 17a-methyltestosterone from the tablet form is affected by interindividual differences in GI motility to a large extent. The MDT data based on the conventional cross-over method are presented in Table IV for comparison. The relative bioavailability data by the conventional method were calculated using the 17a-methyltestosterone tablet data in the stable-isotope coadministration study and the 17a-methyltestosterone solution data in the isotope-effect study on different occasions separated by 1 week. With intra-individual variability in the disposition of
1 Subiect 3
Subject 4
.:
100.
<.
50
(II
Subject 8
i
.-o
10
m
. = 5 U al
0
s
O
1
& 0.5 v)
0
7
8
0
1
-2
3
4
6
Time, h
8
0
1
2
3
4
8
8
0
1
2
3
4
6
8
Time, h
Figure 1-Serum concentration-time cufves for 7 7a-met~yltestosterone(+) and 7 7a-methylte~tosterone-d~ (---H---)after a single oral dose of a 70-mg 7 7a-methyltestosterone tablet and a 70-mg 7 7 ~ - m e t h y l r e s r o s t e - d solution. ~ Journal of Pharmaceutical Sciences / 163 Vol. 75, No. 2, February 7986
Table IlCRelatlve Bloavallablllty of 17rrMethylteatoaterone Tablet by the Stable-Isotope Coadmlnlstratlon Technique
Mean 2
SD
MRT, h
F,,,
Subject
YO
Tablet
Solution
193.9 176.2 134.1 109.6 184.7 401.7 174.6 209.4
192.2 275.0 135.9 97.1 201.8 353.3 139.7 192.3
198.0 88.4
198.4 82.4
Table V-lntra-lndlvldual Varlatlona In the Pharmacoklnetlcs of 17a-Methyltestosterone4E,Solutlon
MDT, h
Tablet
Solution
99.9 63.5 97.8 111.8 90.6 112.5 123.7 107.8
3.36 3.21 2.98 3.59 2.88 2.58 3.32 4.70
2.51 2.07 2.55 2.17 1.56 2.47 2.20 2.36
1.42 1.31 0.12 1.16 2.34
100.9 18.3
3.32 0.64
2.24 0.32
1.09 0.67
Mean f SD
0.85 1.13
0.43
F~I,
MDT, h
83.8 114.1 68.9 101.6 92.7 178.8 69.4 68.5
1.21 1.47 1.23 2.01 0.66 0.47 1.15 2.09
97.2 37.0
1.29 0.57
17a-methyltestosterone, the MDT estimated from the conventional method may be less meaningful. Using the serum concentration-time data obtained from the present stable-isotope coadministration method, the mean AUC was calculated to be 198.0 f 88.4 pmol/ml/h for the 10-mg 17a-methyltestosterone tablet and 198.4 82.4 pmol/mL/h for the 10-mg 17a-methyltestosterone-d3solution (Table 111).The relative bioavailability values (Fre,)ranged from 90.6 to 123.7%in seven of the eight subjects tested. One subject gave a considerably low Frel value of 63.5%, for unknown reasons. Except for this subject, the tablet formulation tested was demonstrated to be equivalent to the oral solution with respect to the extent of absorption. That is, the rate of dissolution did not affect the extent of absorption. We then compared the relative bioavailability obtained by the present stable-isotope coadministration technique with that obtained by the conventional cross-over method (Tables I11 and IV). The mean relative bioavailability among the eight subjects was 97.2% for the conventional method and 100.9% for the stable-isotope coadministration method; the values were practically identical. The CV for the conventional method (38.0%)was -20% higher than that for the stableisotope coadministration method (18.1%), which confirmed the reliability of the stable-isotope coadministration method. The reason for this difference can apparently be attributed to the fundamental assumption of constant clearance in the cross-over method. Significant intra-individual variability in the pharmacokinetic behavior of 17a-methyltestosterone was observed between the two methods of administering 17amethyltestosterone-d3 solution 1 week apart (Table V). For
*
164 / Journal of Pharmaceutical Sciences Vol. 75, No. 2, February 1986
MRT
AUC~~~
Table IV-Relatlve Bloavallablllty of 17a-Methyltestosterone Tablet by the Cross-Over Method
Subject
Week 1Mleek 2
Subject 1 2 3 4 5 6 7 8
~
1.09 0.53 1.41 1.08 1.11 1.65 0.59 0.68
0.86
0.84 0.72 0.70 0.72 1.19 1.06 0.91
example, changes in clearances were indicated by a large deviation in the ratio of the AUC of 17a-methyltestosteroned3 solution in the first week to that in the second week. Therefore, in assessing bioavailability, the assumption of constant clearance in the individual between the occasions of receiving the reference and test dose is suspect for 17amethyltestosterone. The number of subjects required to accurately detect a difference of 20% (a = 0.05, 1 - /3 = 0.8) in the relative bioavailability of two formulations of 17a-methyltestosterone tablets was estimated from the present data according to the method of Heck et al.l2 Although the estimated number of subjects required for a conventional cross-over method was 40,12 subjects were required for the stable-isotope coadministration method.
References and Notes 1. Ruzicka, von L.; Goldgerg, M. W.; Rmenberg, H. R. Helv.Chim. Acta. 1935,18, 1487. 2. Emmens, C. W.; Parkes, A. S. J . End~crinol. 1939,1,323. G. L. Br. Med. J . 1939,2, 11. 3. FOES, 4. Lisser. H.: Curtis. L. E. J . Clin. Endocrinol. 1943.3. 389. 5. Fed. Regist. 1977; 42, 1624. 6. Alkalay, D.; Khemani, L.; Bartlett, M. F. J . Pharm. Sci. 1972, 61. --,1746. -. 7. Alkalay, D.; Khemani, L.; Wagner, W. E.; Bartlett, M. F. J . Clin. Phurm. 1973,13, 142. 8. Baillie, T. A. Phurmacol. Rev. 1981,33, 81. 9. Garland, W. A.; Powell, M. L. J . Chromatogr. Sci. 1981,19,392. 10. Eichelbaum, M.; von Unruh, G. E.;Somogyi, A. Clin.Pharmacokinet. 1982, 7, 490. 11. Murphy, P. J.; Sullivan, H. R. Ann. Rev. P h u r m o l . Taricol. 1980,20, 609. 12. Heck, H. A.; Buttrill, S. E.; Flynn, N. W.; D er, R. L.; Anbar, M.; I
,
Cairns, T.; Dighe, S.; Cabana, B. E. J . $hurmokinet. Biophurm. 1979, 7, 233. 13. Alkalay, D.; Wagner, W. E.; Carlsen, S.; Khemani, L.; Volk, J.; Bartlett, M. F.; LeSher, A. Clin.Pharmacol. Ther. 1980,27,697. 14. Eichelbaum, M.; Dengler, H. J.; Somogyi, A.; von Unruh, G. E. Eur. J . Phurmacol. 1981.19. 127. 15. Shinohara, Y.; Baba, S.yKaauya, Y. Steroids 1984,44, 253. 16. Shinohara, Y.; Baba, S.; Kasuya, Y. J . Chromatogr. 1985,338,
281. 17. Riegelman, S.; Collier, P. J . Phurmacokinet. Biopharm. 1980,8, 509. 18. Gibaldi, M. “Bio harmaceutics and Clinical Pharmacokinetics”; Lea & Febi er: fhiladelphia, 1984; p 135-137. 19. Yamaoka, Nakagawa, T.; Uno, J . Phurmacokinet. Biohurm. 1978,6,547. 20. &anigawara,Y.; Yamaoka, K.; Nakagawa, T.; Uno, T. J . Pharm. Sci. 1982, 71, 1129. 21. Gillespie, W. R.; DiSanto, A. R.; Monovich, R. E.; Albert, K. S . J . Phurm. Sci. 1982, 71,1034. 22. Graffner, C.; Nicklasson, M.; Lindgren, J. J . Phurmacokinet. Biophurm. 1984,12, 367.
k;
f.