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Dissolution of Theophylline from Sustained-Release Dosage Forms and Correlation with Saliva Bioavailability Parameters
Dissolution of Theophylline from Sustained-Release Dosage Forms and Correlation with Saliva Bioavailability Parameters BEE-HWANCHUNG
AND
CHANG-KOO SHIMX
Received February 12, 1987, from the College of Pharmac Seoul National University, Sen 56- 1, Shinlim-Dong, Kwanak-Ku, Seoul 157, Korea. Accepted for publication July 21. 1 9 k . .
~
..
Abstract3 Correlation between in vitro dissolution-character&ics and
in vivo salivary bioavailability parameters of four commercial sustainedrelease and one immediate-release theophylline (TP) or aminophylline dosage forms were examined. Area under the saliva concentration-time , fraction curve up to 6 h (AUC, .&peak saliva concentration (C,,,,,)and absorbed in 1 h (F,),based on saliva concentration following oral administration of TP or aminophylline dosage forms to five volunteers, were closely correlated with percents dissolved in pH 6.8 buffer in 30 min, D30 (6.8),or 60 min, D60 (6.8). Dissolution study in pH 6.8 buffer seemed to be a useful tool for development, evaluation, and quality control of sustained-release dosage forms of TP, since the saliva concentration was reported to represent the blood concentration of TP indirectly. __
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Theophylline (TP) is widely used as a bronchodilator for the treatment of asthma or obstructive pulmonary disease. Sustained-release dosage forms of i t are desirable because of its narrow range of effective blood concentration' and short elimination half-life in humans, especially in children.* Therefore, many sustained-release dosage forms are now available commercially and extensive research- concerning the bioavailability and dissolution characteristics of TP from commercial products have been reported. El-Yazigi and Sawchuk7 found good correlations between bioavailability parameters in the rabbit and dissolution characteristics of TP from sustained-release dosage forms. Brockmeie compared in vitro dissolution rates with in vivo dissolution rates in humans, and Ritschel et al.9 reported correlations between bioavailability parameters in the beagle dog and in vitro dissolution data. But quantitative in vitro-in vivo correlations using human saliva have not been addressed. Good correlation between TP concentrations in blood and saliva has been reported.'&'* The ratio of saliva-to-plasma concentration was confirmed as 0.567 (r = 0.974, n = 90, p < 0.000 001) in our previous studyI3 following iv infusion of aminophylline to 12 healthy male volunteers. The correlation between in vitro dissolution characteristics and in vivo human saliva concentrations of TP from five commercial dosage forms was investigated in this study.
Experimental Section Dosage Forms-Dissolution and saliva bioavailability experiments were performed using the following four commercially available sustained-release dosage forms (A-D) and one immediateA (Slo-bid, lot 54841,300 mg of theophylline release dosage form (E): (TP), William H. Rorer, PA), B (Theolair-SR, lot 27064, 250 mg of TP, Riker Laboratories, CAI, C (Asthcontin, lot 5706, 225 mg of aminophylline, Hyundai Pharmaceutical, Seoul, Korea), D (Theoclear, lot KC-0082, 130 mg of TP, Korea Central Pharmaceutical, Seoul, Korea), and E (Daiwon Aminophylline, lot AMT195, 100 mg of aminophylline, Daiwon Pharmaceutical, Seoul, Korea). Dissolution Studies-Eufers-The dissolution apparatus specified in USP XX (Hanson Research Corp., Northridge, CA) was used. 784 /Journal of Pharmaceutical Sciences Vol. 76, No. 70,October 1987
Dissolution experiments were performed using buffer solutions of pH 1.2 and 6.8, and using a pH change from 1.2 to 6.8. The pH 1.2 buffer was prepared by dissolving 20.0 g of potassium phosphate dibasic in 3 L of distilled water and adjusting to pH 1.2 with hydrochloric acid. The pH 6.8 buffer was prepared by dissolving 20.0 g of potassium phosphate dibasic in 3 L of distilled water and adjusting to pH 6.8 with sodium hydroxide. A programmed pH change from 1.2 (for 2 h) to 6.8 (for 6 h) was also possible automatically. The reagents used for these experiments were of reagent grade. Procedure-Dissolution experiments from four sustained-release dosage forms (A-D) and one immediate-release dosage form (E)were performed for 8 and 3 h, respectively. Three units of each dosage form were subjected to the experiment for the convenience of assay of TP in dissolution fluid. The dissolution fluid (0.9 L)was maintained a t 37 0.5"C. The basket was rotated a t 100 2 1 rpm. The temperature and the rotating speed were maintained throughout this study. Three-milliliter samples were successively taken for TP assay a t 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 h from the dissolution fluids of A, B, C , and D, and a t 15, 30, 45, 60, 75, and 180 min from the dissolution fluids of E. The volume of the fluid removed was replaced by the fresh buffer. The sampling probe of the apparatus was equipped with a 0.22-pm membrane filter. The T P concentration in the sample was determined spectrophotometrically a t 272 nm. Saliva-Rioavailability StudiesSubjects-Five healthy male volunteers, 20-25 years of age, weighing 55-75 kg (mean 63.4 kg) participated in this study. Two of them were light smokers and the others were nonsmokers. After informed consents were obtained, all subjects refrained from the ingestion of coffee, cola, chocolate, and tea for 4 d before and during the study. The subjects were fasted overnight before each dosage and were permitted to eat no food until 3 h after dosing. Smokers were permitted to smoke. Dosing Schedule-A cross-over schedule was designed and a mininum interval of one week was allowed between each dosing. All subjects received a single oral dose of the different dosage forms in each trial with 200 mL of water. Collecting Saliva Samples-Saliva samples were collected at appropriate time intervals up to 24 h. A small amount (20 mg) of citric acid, a salivary flow stimulant, was put on the tongue and held for 1 min. Then, a 2-mL sample of saliva was collected in the test tube and kept frozen until assay. High-Performance Liquid Chromatography Assay of Theophylline in Saliua-The method of Nakano et was modified for TP analysis in saliva. To the 1.0 mL of centrifuged (6000 x g, 5 min) saliva in a glass-stoppered test tube, 50 pL of aqueous solution of phydroxyethyltheophylline (80 pg/mL) was added as an internal standard. After 5 min, 0.1 mI, of 0.1 M HC1 solution was added and the solution was extracted with 7 mL of isopropyla1cohol:chloroform mixture (5:95 viv), after another 5 min, by vortexing (1 min) and centrifugation (1000 x g, 5 min). The aqueous supernatant was aspirated and a 5.0-mL aliquot of the organic layer was evaporated to dryness in a conical tube using a water bath (60 "C) and a stream of dry nitrogen. The residue was redissolved in 200 p L of methanol and a 20-pL aliquot was injected directly onto the column (HewlettPackard, RP-18 10 pm 79915 OD Opp. 174) of a reversed-phase HPLC (Hewlett-Packard, model HP1090) with a UV detector adjusted a t 280 nm. The mobile phase was a 9:91 mixture of acetonitrile: 0.01 M acetate buffer (pH 4.0). The stationary phase was ODS chemically bonded to porous gel (particle size 10 pm) packed in a 20cm stainless steel column (0.46-cm id). The flow rate was 2.0 mL/min
*
0022-3549/'87/1000-0784$01 .OO/O 0 1987, American Pharmaceutical Association
and the mean operating pressure was 1650 psi. The TP concentration in the sample was determined using a calibration curve prepared by the peak height ratio of TP against the internal standard. Bioavailability Parameters-The area under the saliva TP concentration-time curve from time zero to time t (AUCb,) was calculated using the linear trapezoidal rule. The area from time t to infinity was estimated by C,/k, where C, is the saliva TP concentration observed a t time t, and k is the apparent elimination rate constant of TP obtained from the slope of log-linear portion of the curve by least square regression analysis. Moreover, the area from time zero to infinity was calculated bylG
AUCo-, = A U C h ,
+ CJK
(1)
Mean residence time (MRT) of saliva T P concentration after an oral dose was calculated by16
MRT
=
AUMChJAUCk,
(2)
where AUMCo,, is the area under the first moment of the saliva curve from time zero to infinity, which was calculated byL7
AUMCb,
=
AUCo,t + (tC,)/k + CJk
(3)
where AUMCn,, is the AUMC from time zero to time t, and C, is the saliva TP concentration observed a t time t. The AUMC,., was calculated by the trapezoidal rule. Mean absorption time (MAT) of five dosage forms was then obtained bylR
MAT
=
MRT - 1/K
(4)
The fraction of the dose absorbed in time t(F,) was calculated from the Wagner-Nelson equationLY
F,
=
(C, + kAUChtYkAUC-,
(5)
Statistical Analysis-The differences between the percents dissolved from the dosage forms were examined for their significance with the t test. Dose-normalized (i.e., TP 300 mg/60 kg) pharmacokinetic parameters were analyzed using analysis of variance (ANOVA). Any statistical differences found among dosage forms were further compared to determine differences between dosage forms by Turkey's multiple range test. The correlation coefficients of the regression lines between in vitro dissolution parameters and in vivo parameters were obtained by the linear least square method. The correlation coefficients were examined for their significance with the t test.
Results and Discussion
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Figure 1-Release profiles of theophylline in a pH 1.2 dissolution medium. Mean 5 SD values from three experiments. Key to dosage forms: (0) A; (0) B; ( 0 )C; (A) D; (D) E.
The results of the dissolution study in the pH 1.2 buffer solution are shown in Figure 1.Percents of TP or aminophylline dissolved in pH 1.2 buffer from dosage forms A, B, C, and D in 8 h were 80.7 2 3.1, 53.1 2 0.8, 54.4 * 1.3, and 43.9 10.7%, respectively, when expressed as mean 5 SD. More than 95%of aminophylline was dissolved within 1 h for the E dosage form. Percents dissolved from dosage forms B, C, and D were significantly (p < 0.01) smaller than that for the A. Percents dissolved in the pH 6.8 buffer solution from dosage forms A, B, C, and D in 8 h were 82.0 t 1.2, 95.0 5 0.6, 73.8 2 0.8, and 88.3 5 5.6, respectively, as shown in Figure 2. For form E, >95% was dissolved in pH 6.8 buffer within 1 h as in pH 1.2 buffer. Forms B and C showed significant differences (p < 0.01) from A in percents dissolved in 8 h, but D did not. Form A did not show pH-dependent dissolution in pH 1.2 and 6.8 buffers.
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Flgure 2-Release profiles of theophylline in a pH 6.8 dissolution medium. Mean 2 SO values from three experiments. See Figure 7 for key to symbols.
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Figure 3- Logarithmic profiles of percents undissolved in change condition from pH 1.2 (for 2 h) to 6.8 (for 6 h). See Figure 1 for key to symbols. Journal of Pharmaceutical Sciences / 785 Vol. 76, No. 10, October 1987
The pH dependency also appeared when the dissolution study was performed in a pH 1.2 buffer for 2 h, followed by a simultaneous dissolution in pH 6.8 buffer for 6 h (data not shown). The logarithms of percents undissolved versus time were plotted and are shown in Figure 3. The slopes of the lines of B and D changed as the pH of the dissolution fluid changed from 1.2 to 6.8.These results show that the dissolution of TP from slow-release dosage forms is highly dependent on the pH applied. In brief, sustained-release dosage forms, D, B, C, and A, showed pH-dependent dissolution characteristics in that order. The calibration curve of TP in saliva showed good linearity (r = 0.99989)within the range 0.1-4.0 pg/mL. The recovery was 95% and the coefficient of variation over a period of eight . minimum detectable concentration was weeks was ~ 5 %The 0.1 ggimL. Theophylline and /3-hydroxyethyltheophylline, an internal standard, had retention times of 3.97 and 4.93 min, respectively. The metabolites of TP and caffeine (spiked in the saliva sample) were confirmed not to interfere with the quantitation of TP. The saliva concentration of TP following oral administration of dosage form E reached a peak a t 1.30 ? 0.98h and decreased by a one-exponential process. The mean values of the elimination rate constant and half-life were 0.098 h-' and 7.05 h, respectively, and were similar to the reported values.2oSeveral studiesl2.21 showed more rapid elimination
-E
of TP in smokers than in nonsmokers, but two smokers in this study did not show any significant differences in the half-lives of saliva TP from those of three nonsmokers. The half-lives were 6.66 and 7.53 h for smokers, and 6.54,6.86, and 8.06 h for nonsmokers when estimated from the saliva TP curves following administration of immediate-release dosage form E. The saliva concentration of TP following oral administration of five dosage forms were normalized for the dose (i.e., 300 mg TPI6O kg body weight) and are shown in Figure 4. Some saliva bioavailability parameters are listed in Table I. There were significant differences in the peak saliva concentration (C,,,), the time to reach C,, (t,,,), MRT, MAT, and the fraction absorbed in a h ( F 1 )among dosage forms, but not in AUChZ4 and AUCh.,. Some bioavailability parameters showed a good degree of correlation with some dissolution parameters. For example, Figure 5 shows a very high correlation (r = 0.968,p < 0.01) between the fraction absorbed in 1 h ( F 1 )and the percent dissolved in pH 6.8 buffer in 60 min, D60 (6.8). Some pairs of parameters showed significant correlations, as shown in Table 11. The percents of dosage forms dissolved in pH 6.8buffer showed better correlations with some saliva bioavailability parameters (Cmm and AUCh6) than did percents dissolved in pH 1.2 buffer. It was found that some saliva bioavailability parameters, like C,,,, AUCh6, and F1,are correlated significantly with in vitro dissolution data like percent dissolved in pH 6.8buffer in 30 min, D30 (6.8),or that dissolved in 60 min, D60 (6.8).But, D30 (6.8)and D60 (6.8)did not differ significantly in their correlation coefficients with bioavailability parameters. Among three dissolu-
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B C D E Turkey's test ~~
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80
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Figure 4- Dose-normalized (300 mg/60 kg body weight) salivary level profiles of theophylline following administration of five dosage forms. Mean 2 SEM values from five experiments. See Figure 1 for key to symbols. Table I-Dose-Normallzed
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Flgure 5-Correlation between fraction absorbed in 1 h (F,) and percent dissolved in a pH 6.8 dissolution medium in 60 min ID60 (6.8)]; r = 0.968;~ < 0.07.
Parameters following Admlnlstratlon of Flve Dosage Forms
55.25 (5.25) 65.61 (6.39) 63.22 (10.17) 72.69 (21.18) 71.65 (14.47)
73.97 (5.08) 81.14 (11.23) 77.47 (11.52) 84.95 (23.85) 79.79 (14.55)
3.11 (0.25) 4.30 (0.54) 4.35 (0.60) 5.68 (1.45) 7.85 (1.84)
NSb
NS
A
10.0 (1.4) 8.4 (4.6) 4.8 (0.6) 3.4 (0.6) 1.3 (1.0)
A>B>C=-D, -
E
MRT, h
MAT, h
17.67 (0.99) 15.27 (2.13) 14.86 (0.78) 13.08 (0.86) 12.12 (1.25)
7.39 (0.54) 4.99 (1.87) 4.58 (0.81) 2.80 (1.02) 1.83 (1.63)
0.12 (0.06) 0.18 (0.03) 0.24 (0.07) 0.32 (0.16) 0.87 (0.35)
A>& OD> E __ __
A< B
A>B>OD> -
E
~
~
# A = Slo-bid; B = Theolair-SR; C = Asthcontin; D = Theoclear; E = Daiwon aminophyiline; each value represents mean (2SD); n = 5. bNot significant at p = 0.05 level as determined by ANOVA. "Underlined dosage forms are not significantly different from each other (p = 0.05).
786 /Journal of Pharmaceutical Sciences Vol. 76, No. 10, October 1987
Table Il-Correlatlon Coefficlents between Dlssolutlon Parameters and In Vlvo Parameters In Vivo Parameter
Cni, tmax
AUC~B AUCCL-~ MRT" MATd
FI
D30
pH 1,2 0.819' -0.647' 0.700' 0.447' -0.626' 0.627' O.95ag
D60 pH 6.8
0.918g -0.729' 0.827'
0.589' -0.738' 0.740' 0.985'
pH 1.2
pH 6.8
0.797' -0.628' 0.690: 0.421 -0.603' 0.602' 0.951
0.964h -0.767' 0.897O 0.739' -0.837' 0.846' 0.968h
a Percent dissolved in 30 min. Percent dissolved in 60 min. =Mean residence time. dMean absorption time. Fraction absorbed for 1 h. 'Not statistically significant. gp < 0.05.'p < 0.01. 'p < 0.005.
tion fluids of different pH conditions, pH 6.8buffer seemed to reflect C ,,,, AUCC,+~, and F1 better than did pH 1.2 buffer. But, MRT and MAT were not correlated significantly with percents dissolved i n pH 1.2 or 6.8 buffers. In conclusion, it was found that some blood bioavailability parameters of slow-release T P dosage forms, like C,, AUChs, and F1,may be predicted from in vitro dissolution data, like D60 (6.8)or D30 (6.8), by considering the correlation among dissolution, saliva concentration, and blood concentration. It seems convenient and advantageous to use D60 (6.8) or D30 (6.8)instead of blood data, which needs frequent venepuncture for sampling, in t h e development, evaluation, and quality control of T P dosage forms.
3. Weinberger, M.;Hendeles, L.; Wong, L.; Vaughan, L. J . Pediat. 1981,99,145-152. 4. Simons, K . J.; Simons, F. E. R.; Plett, K. D.; Scerbo, C. J . Pharm. Sci. 1984, 73, 939-942. 5. Simons. K. J.: Frith. E. M.: Simons. F. E. R. J . Pharm. Sci. 1982. 71, 505211.' 6. Summers, R. S.;Summers, B.; Rawnsley, S. Znt. J . Pharm. 1986, 30,8348. 7. El-Yazigi, A.; Sawchuk, R. J. J . Pharm. Sci. 1985,74,161-164. 8. Brockmeir, D. Anneim.-Forsch. 1984,34, 1604-1607. 9. Ritschel, W.A.; Koch, H. P.; Alcorn, G. J . Methods Find. Exp. Clin. Pharmacol. 1984,6,609-618. 10. Koysooko, R.; Ellis, E. F.; Levy, G. Clin. Phurmacol. Ther. 1974, 15, 454-460. 11. Shah, V. P.;Riegelman, S. J. Pharm. Sci. 1974,63,1283-1285. 12. Eney, R. D.;Goldstein, E 0. Pediatrics 1976,57,513-517. 13. Park, Kyoung-Ho and co-workers, Abstracts, 35th Annual Convention of the Pharmaceutical Societ of Korea; Pharmaceutical Society of Korea; Seoul, Korea, Octoger, 1986;Abstract 143. 14. Nakano, M.;Nakamura, Y.; Juni, K.; Tomitsuka, T. J . Pharmac o b i e y n . 1980,3, 702-708. 15. Gibaldi, M.;Perrier, D. Pharmacokinetics, Vol. 1; Marcel Dekker: New York. 1975:DD 293-296. 16. Yamaoka, K.; Nakagawa, T.; Uno, T. J . Pharmacokinet. Bioarm. 1978,6,547-558. 17. i t n e t . L.Z.; Galeazzi, R. L. J . Pharm. Sci. 1979,68,1071-1074. 18. Rieaelman, S.; Collier, P. J . Pharmacokinet. Biopharm. 1980.8, 509L534. 19. Wagner, J.G. Fundamentals of Clinical Pharmacokcnetics, 1st Ed.; Dru Intelli ence: Hamilton, IL, 1975;pp 174-176. 20. Myhre, I.; W j s t a d , R. A. Br. J . Clin. Pharmacol. 1983, 15,
k
683-687 - - - - - ..
21. Jenne, J.; Nagasawa, H.; McHugh, R.; MacDonald, F.; Wyse, E. Life Sci. 1975,17, 195-198.
References and Notes 1. Mitenko, P.A,; Ogilvie, R. I. N. Eng. J . Med. 1973,289,600603. 2. Zaske, D. E.; Mitler, K. W.; Strem, E. M.; Austrian, S.;Johnson, P. B. J . Am. Med. Assoc. 1979,237,1453-1455.
Acknowledgments This work was supported by a grant from the Korea Science and Engineering Foundation.
Journal of Pharmaceutical Sciences / 787 Vol. 76,No. 70,October 7987
AND
CHANG-KOO SHIMX
Received February 12, 1987, from the College of Pharmac Seoul National University, Sen 56- 1, Shinlim-Dong, Kwanak-Ku, Seoul 157, Korea. Accepted for publication July 21. 1 9 k . .
~
..
Abstract3 Correlation between in vitro dissolution-character&ics and
in vivo salivary bioavailability parameters of four commercial sustainedrelease and one immediate-release theophylline (TP) or aminophylline dosage forms were examined. Area under the saliva concentration-time , fraction curve up to 6 h (AUC, .&peak saliva concentration (C,,,,,)and absorbed in 1 h (F,),based on saliva concentration following oral administration of TP or aminophylline dosage forms to five volunteers, were closely correlated with percents dissolved in pH 6.8 buffer in 30 min, D30 (6.8),or 60 min, D60 (6.8). Dissolution study in pH 6.8 buffer seemed to be a useful tool for development, evaluation, and quality control of sustained-release dosage forms of TP, since the saliva concentration was reported to represent the blood concentration of TP indirectly. __
_-
.
-
. -.
-
Theophylline (TP) is widely used as a bronchodilator for the treatment of asthma or obstructive pulmonary disease. Sustained-release dosage forms of i t are desirable because of its narrow range of effective blood concentration' and short elimination half-life in humans, especially in children.* Therefore, many sustained-release dosage forms are now available commercially and extensive research- concerning the bioavailability and dissolution characteristics of TP from commercial products have been reported. El-Yazigi and Sawchuk7 found good correlations between bioavailability parameters in the rabbit and dissolution characteristics of TP from sustained-release dosage forms. Brockmeie compared in vitro dissolution rates with in vivo dissolution rates in humans, and Ritschel et al.9 reported correlations between bioavailability parameters in the beagle dog and in vitro dissolution data. But quantitative in vitro-in vivo correlations using human saliva have not been addressed. Good correlation between TP concentrations in blood and saliva has been reported.'&'* The ratio of saliva-to-plasma concentration was confirmed as 0.567 (r = 0.974, n = 90, p < 0.000 001) in our previous studyI3 following iv infusion of aminophylline to 12 healthy male volunteers. The correlation between in vitro dissolution characteristics and in vivo human saliva concentrations of TP from five commercial dosage forms was investigated in this study.
Experimental Section Dosage Forms-Dissolution and saliva bioavailability experiments were performed using the following four commercially available sustained-release dosage forms (A-D) and one immediateA (Slo-bid, lot 54841,300 mg of theophylline release dosage form (E): (TP), William H. Rorer, PA), B (Theolair-SR, lot 27064, 250 mg of TP, Riker Laboratories, CAI, C (Asthcontin, lot 5706, 225 mg of aminophylline, Hyundai Pharmaceutical, Seoul, Korea), D (Theoclear, lot KC-0082, 130 mg of TP, Korea Central Pharmaceutical, Seoul, Korea), and E (Daiwon Aminophylline, lot AMT195, 100 mg of aminophylline, Daiwon Pharmaceutical, Seoul, Korea). Dissolution Studies-Eufers-The dissolution apparatus specified in USP XX (Hanson Research Corp., Northridge, CA) was used. 784 /Journal of Pharmaceutical Sciences Vol. 76, No. 70,October 1987
Dissolution experiments were performed using buffer solutions of pH 1.2 and 6.8, and using a pH change from 1.2 to 6.8. The pH 1.2 buffer was prepared by dissolving 20.0 g of potassium phosphate dibasic in 3 L of distilled water and adjusting to pH 1.2 with hydrochloric acid. The pH 6.8 buffer was prepared by dissolving 20.0 g of potassium phosphate dibasic in 3 L of distilled water and adjusting to pH 6.8 with sodium hydroxide. A programmed pH change from 1.2 (for 2 h) to 6.8 (for 6 h) was also possible automatically. The reagents used for these experiments were of reagent grade. Procedure-Dissolution experiments from four sustained-release dosage forms (A-D) and one immediate-release dosage form (E)were performed for 8 and 3 h, respectively. Three units of each dosage form were subjected to the experiment for the convenience of assay of TP in dissolution fluid. The dissolution fluid (0.9 L)was maintained a t 37 0.5"C. The basket was rotated a t 100 2 1 rpm. The temperature and the rotating speed were maintained throughout this study. Three-milliliter samples were successively taken for TP assay a t 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 h from the dissolution fluids of A, B, C , and D, and a t 15, 30, 45, 60, 75, and 180 min from the dissolution fluids of E. The volume of the fluid removed was replaced by the fresh buffer. The sampling probe of the apparatus was equipped with a 0.22-pm membrane filter. The T P concentration in the sample was determined spectrophotometrically a t 272 nm. Saliva-Rioavailability StudiesSubjects-Five healthy male volunteers, 20-25 years of age, weighing 55-75 kg (mean 63.4 kg) participated in this study. Two of them were light smokers and the others were nonsmokers. After informed consents were obtained, all subjects refrained from the ingestion of coffee, cola, chocolate, and tea for 4 d before and during the study. The subjects were fasted overnight before each dosage and were permitted to eat no food until 3 h after dosing. Smokers were permitted to smoke. Dosing Schedule-A cross-over schedule was designed and a mininum interval of one week was allowed between each dosing. All subjects received a single oral dose of the different dosage forms in each trial with 200 mL of water. Collecting Saliva Samples-Saliva samples were collected at appropriate time intervals up to 24 h. A small amount (20 mg) of citric acid, a salivary flow stimulant, was put on the tongue and held for 1 min. Then, a 2-mL sample of saliva was collected in the test tube and kept frozen until assay. High-Performance Liquid Chromatography Assay of Theophylline in Saliua-The method of Nakano et was modified for TP analysis in saliva. To the 1.0 mL of centrifuged (6000 x g, 5 min) saliva in a glass-stoppered test tube, 50 pL of aqueous solution of phydroxyethyltheophylline (80 pg/mL) was added as an internal standard. After 5 min, 0.1 mI, of 0.1 M HC1 solution was added and the solution was extracted with 7 mL of isopropyla1cohol:chloroform mixture (5:95 viv), after another 5 min, by vortexing (1 min) and centrifugation (1000 x g, 5 min). The aqueous supernatant was aspirated and a 5.0-mL aliquot of the organic layer was evaporated to dryness in a conical tube using a water bath (60 "C) and a stream of dry nitrogen. The residue was redissolved in 200 p L of methanol and a 20-pL aliquot was injected directly onto the column (HewlettPackard, RP-18 10 pm 79915 OD Opp. 174) of a reversed-phase HPLC (Hewlett-Packard, model HP1090) with a UV detector adjusted a t 280 nm. The mobile phase was a 9:91 mixture of acetonitrile: 0.01 M acetate buffer (pH 4.0). The stationary phase was ODS chemically bonded to porous gel (particle size 10 pm) packed in a 20cm stainless steel column (0.46-cm id). The flow rate was 2.0 mL/min
*
0022-3549/'87/1000-0784$01 .OO/O 0 1987, American Pharmaceutical Association
and the mean operating pressure was 1650 psi. The TP concentration in the sample was determined using a calibration curve prepared by the peak height ratio of TP against the internal standard. Bioavailability Parameters-The area under the saliva TP concentration-time curve from time zero to time t (AUCb,) was calculated using the linear trapezoidal rule. The area from time t to infinity was estimated by C,/k, where C, is the saliva TP concentration observed a t time t, and k is the apparent elimination rate constant of TP obtained from the slope of log-linear portion of the curve by least square regression analysis. Moreover, the area from time zero to infinity was calculated bylG
AUCo-, = A U C h ,
+ CJK
(1)
Mean residence time (MRT) of saliva T P concentration after an oral dose was calculated by16
MRT
=
AUMChJAUCk,
(2)
where AUMCo,, is the area under the first moment of the saliva curve from time zero to infinity, which was calculated byL7
AUMCb,
=
AUCo,t + (tC,)/k + CJk
(3)
where AUMCn,, is the AUMC from time zero to time t, and C, is the saliva TP concentration observed a t time t. The AUMC,., was calculated by the trapezoidal rule. Mean absorption time (MAT) of five dosage forms was then obtained bylR
MAT
=
MRT - 1/K
(4)
The fraction of the dose absorbed in time t(F,) was calculated from the Wagner-Nelson equationLY
F,
=
(C, + kAUChtYkAUC-,
(5)
Statistical Analysis-The differences between the percents dissolved from the dosage forms were examined for their significance with the t test. Dose-normalized (i.e., TP 300 mg/60 kg) pharmacokinetic parameters were analyzed using analysis of variance (ANOVA). Any statistical differences found among dosage forms were further compared to determine differences between dosage forms by Turkey's multiple range test. The correlation coefficients of the regression lines between in vitro dissolution parameters and in vivo parameters were obtained by the linear least square method. The correlation coefficients were examined for their significance with the t test.
Results and Discussion
I
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,
6
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Figure 1-Release profiles of theophylline in a pH 1.2 dissolution medium. Mean 5 SD values from three experiments. Key to dosage forms: (0) A; (0) B; ( 0 )C; (A) D; (D) E.
The results of the dissolution study in the pH 1.2 buffer solution are shown in Figure 1.Percents of TP or aminophylline dissolved in pH 1.2 buffer from dosage forms A, B, C, and D in 8 h were 80.7 2 3.1, 53.1 2 0.8, 54.4 * 1.3, and 43.9 10.7%, respectively, when expressed as mean 5 SD. More than 95%of aminophylline was dissolved within 1 h for the E dosage form. Percents dissolved from dosage forms B, C, and D were significantly (p < 0.01) smaller than that for the A. Percents dissolved in the pH 6.8 buffer solution from dosage forms A, B, C, and D in 8 h were 82.0 t 1.2, 95.0 5 0.6, 73.8 2 0.8, and 88.3 5 5.6, respectively, as shown in Figure 2. For form E, >95% was dissolved in pH 6.8 buffer within 1 h as in pH 1.2 buffer. Forms B and C showed significant differences (p < 0.01) from A in percents dissolved in 8 h, but D did not. Form A did not show pH-dependent dissolution in pH 1.2 and 6.8 buffers.
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Flgure 2-Release profiles of theophylline in a pH 6.8 dissolution medium. Mean 2 SO values from three experiments. See Figure 7 for key to symbols.
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Figure 3- Logarithmic profiles of percents undissolved in change condition from pH 1.2 (for 2 h) to 6.8 (for 6 h). See Figure 1 for key to symbols. Journal of Pharmaceutical Sciences / 785 Vol. 76, No. 10, October 1987
The pH dependency also appeared when the dissolution study was performed in a pH 1.2 buffer for 2 h, followed by a simultaneous dissolution in pH 6.8 buffer for 6 h (data not shown). The logarithms of percents undissolved versus time were plotted and are shown in Figure 3. The slopes of the lines of B and D changed as the pH of the dissolution fluid changed from 1.2 to 6.8.These results show that the dissolution of TP from slow-release dosage forms is highly dependent on the pH applied. In brief, sustained-release dosage forms, D, B, C, and A, showed pH-dependent dissolution characteristics in that order. The calibration curve of TP in saliva showed good linearity (r = 0.99989)within the range 0.1-4.0 pg/mL. The recovery was 95% and the coefficient of variation over a period of eight . minimum detectable concentration was weeks was ~ 5 %The 0.1 ggimL. Theophylline and /3-hydroxyethyltheophylline, an internal standard, had retention times of 3.97 and 4.93 min, respectively. The metabolites of TP and caffeine (spiked in the saliva sample) were confirmed not to interfere with the quantitation of TP. The saliva concentration of TP following oral administration of dosage form E reached a peak a t 1.30 ? 0.98h and decreased by a one-exponential process. The mean values of the elimination rate constant and half-life were 0.098 h-' and 7.05 h, respectively, and were similar to the reported values.2oSeveral studiesl2.21 showed more rapid elimination
-E
of TP in smokers than in nonsmokers, but two smokers in this study did not show any significant differences in the half-lives of saliva TP from those of three nonsmokers. The half-lives were 6.66 and 7.53 h for smokers, and 6.54,6.86, and 8.06 h for nonsmokers when estimated from the saliva TP curves following administration of immediate-release dosage form E. The saliva concentration of TP following oral administration of five dosage forms were normalized for the dose (i.e., 300 mg TPI6O kg body weight) and are shown in Figure 4. Some saliva bioavailability parameters are listed in Table I. There were significant differences in the peak saliva concentration (C,,,), the time to reach C,, (t,,,), MRT, MAT, and the fraction absorbed in a h ( F 1 )among dosage forms, but not in AUChZ4 and AUCh.,. Some bioavailability parameters showed a good degree of correlation with some dissolution parameters. For example, Figure 5 shows a very high correlation (r = 0.968,p < 0.01) between the fraction absorbed in 1 h ( F 1 )and the percent dissolved in pH 6.8 buffer in 60 min, D60 (6.8). Some pairs of parameters showed significant correlations, as shown in Table 11. The percents of dosage forms dissolved in pH 6.8buffer showed better correlations with some saliva bioavailability parameters (Cmm and AUCh6) than did percents dissolved in pH 1.2 buffer. It was found that some saliva bioavailability parameters, like C,,,, AUCh6, and F1,are correlated significantly with in vitro dissolution data like percent dissolved in pH 6.8buffer in 30 min, D30 (6.8),or that dissolved in 60 min, D60 (6.8).But, D30 (6.8)and D60 (6.8)did not differ significantly in their correlation coefficients with bioavailability parameters. Among three dissolu-
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Figure 4- Dose-normalized (300 mg/60 kg body weight) salivary level profiles of theophylline following administration of five dosage forms. Mean 2 SEM values from five experiments. See Figure 1 for key to symbols. Table I-Dose-Normallzed
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Flgure 5-Correlation between fraction absorbed in 1 h (F,) and percent dissolved in a pH 6.8 dissolution medium in 60 min ID60 (6.8)]; r = 0.968;~ < 0.07.
Parameters following Admlnlstratlon of Flve Dosage Forms
55.25 (5.25) 65.61 (6.39) 63.22 (10.17) 72.69 (21.18) 71.65 (14.47)
73.97 (5.08) 81.14 (11.23) 77.47 (11.52) 84.95 (23.85) 79.79 (14.55)
3.11 (0.25) 4.30 (0.54) 4.35 (0.60) 5.68 (1.45) 7.85 (1.84)
NSb
NS
A
10.0 (1.4) 8.4 (4.6) 4.8 (0.6) 3.4 (0.6) 1.3 (1.0)
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MRT, h
MAT, h
17.67 (0.99) 15.27 (2.13) 14.86 (0.78) 13.08 (0.86) 12.12 (1.25)
7.39 (0.54) 4.99 (1.87) 4.58 (0.81) 2.80 (1.02) 1.83 (1.63)
0.12 (0.06) 0.18 (0.03) 0.24 (0.07) 0.32 (0.16) 0.87 (0.35)
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# A = Slo-bid; B = Theolair-SR; C = Asthcontin; D = Theoclear; E = Daiwon aminophyiline; each value represents mean (2SD); n = 5. bNot significant at p = 0.05 level as determined by ANOVA. "Underlined dosage forms are not significantly different from each other (p = 0.05).
786 /Journal of Pharmaceutical Sciences Vol. 76, No. 10, October 1987
Table Il-Correlatlon Coefficlents between Dlssolutlon Parameters and In Vlvo Parameters In Vivo Parameter
Cni, tmax
AUC~B AUCCL-~ MRT" MATd
FI
D30
pH 1,2 0.819' -0.647' 0.700' 0.447' -0.626' 0.627' O.95ag
D60 pH 6.8
0.918g -0.729' 0.827'
0.589' -0.738' 0.740' 0.985'
pH 1.2
pH 6.8
0.797' -0.628' 0.690: 0.421 -0.603' 0.602' 0.951
0.964h -0.767' 0.897O 0.739' -0.837' 0.846' 0.968h
a Percent dissolved in 30 min. Percent dissolved in 60 min. =Mean residence time. dMean absorption time. Fraction absorbed for 1 h. 'Not statistically significant. gp < 0.05.'p < 0.01. 'p < 0.005.
tion fluids of different pH conditions, pH 6.8buffer seemed to reflect C ,,,, AUCC,+~, and F1 better than did pH 1.2 buffer. But, MRT and MAT were not correlated significantly with percents dissolved i n pH 1.2 or 6.8 buffers. In conclusion, it was found that some blood bioavailability parameters of slow-release T P dosage forms, like C,, AUChs, and F1,may be predicted from in vitro dissolution data, like D60 (6.8)or D30 (6.8), by considering the correlation among dissolution, saliva concentration, and blood concentration. It seems convenient and advantageous to use D60 (6.8) or D30 (6.8)instead of blood data, which needs frequent venepuncture for sampling, in t h e development, evaluation, and quality control of T P dosage forms.
3. Weinberger, M.;Hendeles, L.; Wong, L.; Vaughan, L. J . Pediat. 1981,99,145-152. 4. Simons, K . J.; Simons, F. E. R.; Plett, K. D.; Scerbo, C. J . Pharm. Sci. 1984, 73, 939-942. 5. Simons. K. J.: Frith. E. M.: Simons. F. E. R. J . Pharm. Sci. 1982. 71, 505211.' 6. Summers, R. S.;Summers, B.; Rawnsley, S. Znt. J . Pharm. 1986, 30,8348. 7. El-Yazigi, A.; Sawchuk, R. J. J . Pharm. Sci. 1985,74,161-164. 8. Brockmeir, D. Anneim.-Forsch. 1984,34, 1604-1607. 9. Ritschel, W.A.; Koch, H. P.; Alcorn, G. J . Methods Find. Exp. Clin. Pharmacol. 1984,6,609-618. 10. Koysooko, R.; Ellis, E. F.; Levy, G. Clin. Phurmacol. Ther. 1974, 15, 454-460. 11. Shah, V. P.;Riegelman, S. J. Pharm. Sci. 1974,63,1283-1285. 12. Eney, R. D.;Goldstein, E 0. Pediatrics 1976,57,513-517. 13. Park, Kyoung-Ho and co-workers, Abstracts, 35th Annual Convention of the Pharmaceutical Societ of Korea; Pharmaceutical Society of Korea; Seoul, Korea, Octoger, 1986;Abstract 143. 14. Nakano, M.;Nakamura, Y.; Juni, K.; Tomitsuka, T. J . Pharmac o b i e y n . 1980,3, 702-708. 15. Gibaldi, M.;Perrier, D. Pharmacokinetics, Vol. 1; Marcel Dekker: New York. 1975:DD 293-296. 16. Yamaoka, K.; Nakagawa, T.; Uno, T. J . Pharmacokinet. Bioarm. 1978,6,547-558. 17. i t n e t . L.Z.; Galeazzi, R. L. J . Pharm. Sci. 1979,68,1071-1074. 18. Rieaelman, S.; Collier, P. J . Pharmacokinet. Biopharm. 1980.8, 509L534. 19. Wagner, J.G. Fundamentals of Clinical Pharmacokcnetics, 1st Ed.; Dru Intelli ence: Hamilton, IL, 1975;pp 174-176. 20. Myhre, I.; W j s t a d , R. A. Br. J . Clin. Pharmacol. 1983, 15,
k
683-687 - - - - - ..
21. Jenne, J.; Nagasawa, H.; McHugh, R.; MacDonald, F.; Wyse, E. Life Sci. 1975,17, 195-198.
References and Notes 1. Mitenko, P.A,; Ogilvie, R. I. N. Eng. J . Med. 1973,289,600603. 2. Zaske, D. E.; Mitler, K. W.; Strem, E. M.; Austrian, S.;Johnson, P. B. J . Am. Med. Assoc. 1979,237,1453-1455.
Acknowledgments This work was supported by a grant from the Korea Science and Engineering Foundation.
Journal of Pharmaceutical Sciences / 787 Vol. 76,No. 70,October 7987