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Application of Accelerated Testing to Shelf-life Prediction of Commercial Protein Preparations
COMMUNICATIONS
Application of Accelerated Testing to Shelf-life Prediction of Commercial Protein Preparations To the Editor: It is believed that stability predictions based on the Arrhenius relationship are inappropriate for protein preparations which exhibit complex degradation mechanisms. The degradation mechanisms of some protein drugs have been reported to vary as a function of temperature.' Extrapolation of stability data obtained with such protein drugs to lower temperatures should be limited to the temperature range over which the same degradation pathway is operative. Further, it has been suggested that Arrhenius approach should be conducted in the temperature range over which protein drugs are not susceptible to unfolding, usually below 40 "C.2 However, there have been too few papers concerning degradation kinetics and Arrhenius behavior of protein drugs to determine the valid temperature range for stability studies. Several papers have described the Arrhenius plots for chemical degradation pathways such as deamidation,3p4hydrolysis,5,6and racemi~ation~ of peptides. The Arrhenius plots for inactivation of some proteins during storage have also been reported, even though the inactivation mechanismswere unknown. Inactivation during storage of dry horse serum cholinesterase*and lyophilized human interferon-@gexhibited linear Arrhenius plots in the ranges of 38-140 "C and of 25-80 "C, respectively. Linear Arrhenius plots were also observed for the solid-state inactivation of digestive enzymes such as Rhizopus lipase (45-60 "C) and pancreatic lipase and a-amylase (40-55 "C). The activation energies were calculated from the slopes of the lines to be 23-58 kcal/mol."J Inactivation of lyophilized urokinase showed linear Arrhenius plots from 30 to 50 "C and gave an activation energy of 15 kcalfmol.1' The present study was carried out to obtain further information on the Arrhenius behavior of protein drug degradation. Commercial preparations of enzymes for medicinal use (achymotrypsin troche and tablet, bromelain tablet, kallikrein capsule, and P-galactosidasepowder) were used as model protein preparations, and the inactivation during storage of these preparations was studied as a function of temperature. Stability Studies-Commercial a-chymotrypsin troch, a-chymotrypsin tablet, bromelain tablets, kallikrein capsule, and @-galactosidasepowder were purchased from the manufacturers and stored under degradation-accelerating conditions [40-70 "C, 50 or 75% relative humidity (RH)]. The outer packages of these preparations were removed, leaving the formulation enclosed in the primary package. Relative humiditywas adjusted with NaBr (50% RH) or NaCl (75% RH) saturated solutions. Preparations were also stored a t 25 "C and 50 or 75% RH for long-term stability studies. Samples were taken from the stored preparations at appropriate intervals for assay of activity. a-Chymotrypsin was extracted by crushing troche and tablet preparations in adequate volumes of 0.001 N HCl solution. The coating film of the tablets had been removed with phosphate buffer solution (pH 7.0, 50 mM) prior to the extraction. The a-chymotrypsin solution was centrifuged and the supernate was diluted with 0.001 N HCl to yield an activity unit of about 1.5 pmol N-acetyl-L-tyrosinethyl ester (ATEE, Aldrich, Milwaukee, WI)/min/mL. The activity of the sample solution was determined by the USP method,12 using ATEE. Bromelain was extracted with a pH 4.5 solution containing 5.27 mg/mL cystein, 2.23 mg/mL EDTA, and 23.4 mg/mL NaC1, 454 /Journal of Pharmaceutical Sciences Vol. 83, No. 3, March 1994
as described by Tanimoto et al.13 The samplesolution was diluted with the same cystein solution to give an activity unit of about 50 pg tyrosine/min/mL. The activity was determined by the method described in a previous paper.14 Kallikrein was extracted with 100 mM tris(hydroxymethy1)aminomethane solution (pH 8.0) to give an activity unit of about 5 pmol benzoyl-L-arginine (Tokyo Kasei Co., Tokyo, Japan)/ min/mL. The activity was measured by the high-performance liquid chromatographic method using benzoyl-L-arginineethyl ester hydrochloride (Sigma, St. Louis, MO) as described previo~sly.~~ The extraction procedures of a-chymotrypsin, bromelain, and kallikrein from preparations, used in the present study, have been generally employed as standard methods. P-Galactosidase powder preparation was dissolved in distilled water to yield an activity unit of about 0.1 pmol o-nitrophenol/ min/mL. The activity measurement was carried out using 2-nitrophenyl P-D-gdactopyranoside (Wako Chemical Industry Co., Osaka, Japan) as a substrate by the method described in a previous paper.15 Estimation of Time Required for 10% Inactivation (tgO)-Remaining activity ( x ) - time ( t ) data observed under accelerated degradation conditions were fitted by nonlinear regression analysis according to a triexponential equation (eq l), except for the inactivation of bromelain tablet A, which was fitted according to a parabolic equation (eq 2) x = a exp(bt)
+ c exp(dt) + (1 - a - c)exp(et)
(1)
+ +
x = a bt C t 2 (2) where a-e are constants. The t w for each accelerated condition was calculated from the regression curve obtained. The x - t data observed a t 25 "C were treated by Woolfe's equation16to calculate the tw a t 25 "C and the 95% confidence interval. Figure 1shows the typical activity remaining-time profiles of a-chymotrypsin troche, a-chymotrypsin tablet, bromelain tablets A and B, kallikrein capsule, and 8-galactosidase powder under accelerated degradation conditions. The solid lines in the figure show nonlinear regression curves. Each preparation revealed a different complex profile and no theoretical kinetics could be applied to calculate kinetic parameters. The complexity of the observed profiles may possibly result from the heterogeneity of degradative reactions in the solid state and/or complex kinetics of protein degradation. It is known that heterogeneous degradation of drugs in solid dosage forms often exhibits complex profiles of a similar nature to those observed here. On the other hand, kinetic studies have shown that protein degradation may conform to various kinetic m0dels.~llJ~J7 In the present study bromelain tablets A and B showed different types of degradation curves, since one fit a parabolic curve while the other fit exponential kinetics. The difference in the degradation profile of bromelain between tablets suggests that the complex degradation time courses observed for the preparations studied reflects the heterogeneous solid-state degradation behavior rather than complex protein degradation kinetics. The reciprocal of the time required for 10% inactivation, l/tw, was calculated from the nonlinear regression curve and used as a measure of the inactivation rate.
0022-3549/94/ 1200-454$04.50/0
0 1994, American Chemical Society and American Pharmaceutical Association
4
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,
.
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. 800
600
.
1
.
1000
,
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3.2
3.0
Time ( h r )
Flgure 1-Inacthration of a-chymottypsintroche ( 0 )and tablet ('I at) 60 and 75% RH, bromelain tablets A (A)and B (0)at 50 *Cand 75% RH, kallikrein capsule at 55 O C and 75% RH (O),and 0-galactosidase powder at 60 O C and 50% RH (A). OC
3.0
3.2 1000/T
3.4
(K-l)
Flgure 3-The effect of temperature on inactivation of a-chymottypsin troch (A),a-chymotrypsintablet (A),/%gaabctosidasepowder (O),bromelaln tablet A (V),bromelain tablet B (V),and kallikrein capsule (0).
Table 1-Shelf-Lives
of Commercial Protein Preparations at 25
"c (f00,25)
100
-
3.4
1000/T ( K - l )
confidence interval) apparent Observed estimateda €ab (month) (month) (kcai/moi)
f90,25 (95 % 80
Preparation
2
M
Iga
a-Chymotrypsin troche a-Chymotrypsin tablet Brornelain tablet A Bromeiain tablet B Kallikrein capsule P-Galactosidase powder
60
E
E
A
X .* +
40
8 0
. . . . .I . ........I . . .
I......-...
0 0
100
200
300 Time (hr)
Time
400
8.0 (3.5-12.5) 11.2 (5.7-17.0) 7.1 (2.4-11.7) 4.2 (0.8-7.4) 0.8 (0.2-1.3) 4.3 (2.2-6.5)
5.1 (2.1-12.1) 9.4 (0.5-171) 6.0(2.4-15.0) 1.7 (0.6-5.1) 0.7 (0.6-0.8) 2.9 (0.03-241)
15.6 15.6 11.6 13.5 12.1 17.9
Calculated from the data obtained at accelerated conditions. Calculated from the data obtained at 25 "C and under accelerated degradation conditions. ......I.........,
500
600
(month)
Flgure 2-Inactivation of a-chymotrypsintroche at 50 OC (A), 60 OC (A), and 70 O C (0)(a)and at 25 "C(0)(b). RH was 75 YO. Solid lines represent regression curve and 95% confidence intervals.
Figures 2 shows the remaining activity-time profiles of the a-chymotrypsin troche observed at elevated temperatures ( a ) and that observed at 25 "C (b). The degradation up to 10-20% at 25 "C could be fitted by a linear regression curve. Similar curves were observed for the other preparations. The tw and 95% confidence interval calculated by Woolfe's equation for each preparation are shown in Table 1. Figure 3 shows the Arrhenius plots for the l / t w observed at each temperature. The solid lines were calculated by linearregression analysis. An approximately linear relationship was observed in the temperature range studied for the preparations. The apparent activation energy was calculated from the slope of each line, and these are shown in Table 1.
The tw at 25 "C was estimated from the Arrhenius plots of the data obtained at accelerated degradation conditions above 40 "C for each preparation, and these values and 95 % confidence intervals are shown in Table 1. All the estimates fell within the 95% confidence intervals of the tw calculated from the inactivation data at 25 "C. For some preparations, however, a significant difference was observed between the estimated and observed tw. The bromelain tablet B was the typical case. The tw of this preparation estimated from the accelerated data was much smaller than that observed at 25 "C, indicating that the activation energy in the temperature range above 40 "C tends to be smaller than that in lower temperature range for this preparation. It is often the case for degradation of solid-dosageforms (even for a drug of a small molecular weight) that the Arrhenius plots deviate from a linear relationship. This can be ascribed to the change of a rate-determining step with temperature in heterogeneous reactions (e.g. change from water absorption step to chemical breakdown step). The small degree of deviation observed in the Arrhenius plots for the inactivation of the protein preparations studied may thus be due to the heterogeneity of the solid system rather than to the fit of protein degradation to Arrhenius behavior per se. The t w estimated from the accelerated data of the a-chymotrypsin tablet and 0-galactosidasepowder provided large 95 3'% confidence intervals, since the Arrhenius plots of these accelerated data were not linear. This indicates that accelerated Journal of Pharmaceutical Sciences / 455 Vol. 83, No. 3, March 1994
degradation testing has to be carefully designed regarding the number of temperature levels, etc., in order to get reliable tw estimates. In conclusion, though the inactivation of six Protein PreParations studied showed complex kinetics, the reciprocal of the tw, a measure of inactivation rate, exhibited an approximately linear Arrhenius relationship. The resultsindicate that it may be reasonable in gome cases to extrapolate the results obtained at higher temperature to predict the shelf-life of protein preparations.
References and Notes 1. Gu, K. M.; Erdos, E. A.; Chian , H.; Calderwood, T.; Tsai, K.; Visor, G. C.; Duffy, J.; Hsu, W.; jester, L. C. Pharm. Res. 1991,8, 485-490. 2. Pearlman, R.;Nguyen, T. J. Pharm. Pharmacol. 1992,44(Suppl. l), 178-185. 3. Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7,703-711. 4. Patel, K.; Borchardt, R. T. Pharm. Res. 1990,787-793. 5. Helm, V. J.; Muller, B. W. Pharm. Res. 1990,1253-1256. 6. Motto, M.G.; Hamburg, P. F.; Graden, D. A.; Shaw, C. J.; Cotter, M. L. J. Pharm. Sci. 1991,80,419-423. 7. Friedman, M.; Masters, P. M. J. Food Sci. 1982,47, 760-764. 8. Cole, B.R.; Leadbeater, L. J. Pharm. Pharmacol. 1968,20,48-53.
456 / Journal of Pharmaceutical Sciences Vol. 83. No. 3, March 1994
9. Geigert, J.; Ziegler, D. L.; Panschar, B. M.; Creasey, A. A.; Vitt, C. R. J. Interferon Res. 1987,7, 203-211. 10. Sugiura, M.; Kurobe, M.; Tamura, S.; Ikeda, S. Chem.Pharm.Bull. 1981,29,2096-2100. 11. Patel, J. P.Drug. Dev. Ind. Pharm. 1990,16,2613-2626. 12. U.S. Pharmacopia XXZZ, 1990. 13. Tmimoto, T. GekkanYakuji 1983,259 1183-1188. 14. Yoshioka, s-;IZutSU, K.; h 0 , y.; Takeda, y. Pharm. Res. 1991.8, 480-484. 15. Izutsu, K.;Yoshioka, S.; Takeda, Y. Chem. Pharm. Bull. 199O,s, 800-803. 16. Woolfe, A. J.; Worthington, H. E. C. Drug Dev. Commun. 19741975,1,185-210. 17. Ahern, T.J.; Kilibanov, A. M. Science 1985,228,1280-1284.
SUMIE YOSHIOKA~,YUKIO Aso, KEN-ICHI IZUTSU, AND TADAOTERAO National Institute of Health Sciences 1-18-1, Kamlyoga, Setagaya-ku Tokyo 158, Japan Received November 5, 1992. Accepted for publication December 27, 1993.
Application of Accelerated Testing to Shelf-life Prediction of Commercial Protein Preparations To the Editor: It is believed that stability predictions based on the Arrhenius relationship are inappropriate for protein preparations which exhibit complex degradation mechanisms. The degradation mechanisms of some protein drugs have been reported to vary as a function of temperature.' Extrapolation of stability data obtained with such protein drugs to lower temperatures should be limited to the temperature range over which the same degradation pathway is operative. Further, it has been suggested that Arrhenius approach should be conducted in the temperature range over which protein drugs are not susceptible to unfolding, usually below 40 "C.2 However, there have been too few papers concerning degradation kinetics and Arrhenius behavior of protein drugs to determine the valid temperature range for stability studies. Several papers have described the Arrhenius plots for chemical degradation pathways such as deamidation,3p4hydrolysis,5,6and racemi~ation~ of peptides. The Arrhenius plots for inactivation of some proteins during storage have also been reported, even though the inactivation mechanismswere unknown. Inactivation during storage of dry horse serum cholinesterase*and lyophilized human interferon-@gexhibited linear Arrhenius plots in the ranges of 38-140 "C and of 25-80 "C, respectively. Linear Arrhenius plots were also observed for the solid-state inactivation of digestive enzymes such as Rhizopus lipase (45-60 "C) and pancreatic lipase and a-amylase (40-55 "C). The activation energies were calculated from the slopes of the lines to be 23-58 kcal/mol."J Inactivation of lyophilized urokinase showed linear Arrhenius plots from 30 to 50 "C and gave an activation energy of 15 kcalfmol.1' The present study was carried out to obtain further information on the Arrhenius behavior of protein drug degradation. Commercial preparations of enzymes for medicinal use (achymotrypsin troche and tablet, bromelain tablet, kallikrein capsule, and P-galactosidasepowder) were used as model protein preparations, and the inactivation during storage of these preparations was studied as a function of temperature. Stability Studies-Commercial a-chymotrypsin troch, a-chymotrypsin tablet, bromelain tablets, kallikrein capsule, and @-galactosidasepowder were purchased from the manufacturers and stored under degradation-accelerating conditions [40-70 "C, 50 or 75% relative humidity (RH)]. The outer packages of these preparations were removed, leaving the formulation enclosed in the primary package. Relative humiditywas adjusted with NaBr (50% RH) or NaCl (75% RH) saturated solutions. Preparations were also stored a t 25 "C and 50 or 75% RH for long-term stability studies. Samples were taken from the stored preparations at appropriate intervals for assay of activity. a-Chymotrypsin was extracted by crushing troche and tablet preparations in adequate volumes of 0.001 N HCl solution. The coating film of the tablets had been removed with phosphate buffer solution (pH 7.0, 50 mM) prior to the extraction. The a-chymotrypsin solution was centrifuged and the supernate was diluted with 0.001 N HCl to yield an activity unit of about 1.5 pmol N-acetyl-L-tyrosinethyl ester (ATEE, Aldrich, Milwaukee, WI)/min/mL. The activity of the sample solution was determined by the USP method,12 using ATEE. Bromelain was extracted with a pH 4.5 solution containing 5.27 mg/mL cystein, 2.23 mg/mL EDTA, and 23.4 mg/mL NaC1, 454 /Journal of Pharmaceutical Sciences Vol. 83, No. 3, March 1994
as described by Tanimoto et al.13 The samplesolution was diluted with the same cystein solution to give an activity unit of about 50 pg tyrosine/min/mL. The activity was determined by the method described in a previous paper.14 Kallikrein was extracted with 100 mM tris(hydroxymethy1)aminomethane solution (pH 8.0) to give an activity unit of about 5 pmol benzoyl-L-arginine (Tokyo Kasei Co., Tokyo, Japan)/ min/mL. The activity was measured by the high-performance liquid chromatographic method using benzoyl-L-arginineethyl ester hydrochloride (Sigma, St. Louis, MO) as described previo~sly.~~ The extraction procedures of a-chymotrypsin, bromelain, and kallikrein from preparations, used in the present study, have been generally employed as standard methods. P-Galactosidase powder preparation was dissolved in distilled water to yield an activity unit of about 0.1 pmol o-nitrophenol/ min/mL. The activity measurement was carried out using 2-nitrophenyl P-D-gdactopyranoside (Wako Chemical Industry Co., Osaka, Japan) as a substrate by the method described in a previous paper.15 Estimation of Time Required for 10% Inactivation (tgO)-Remaining activity ( x ) - time ( t ) data observed under accelerated degradation conditions were fitted by nonlinear regression analysis according to a triexponential equation (eq l), except for the inactivation of bromelain tablet A, which was fitted according to a parabolic equation (eq 2) x = a exp(bt)
+ c exp(dt) + (1 - a - c)exp(et)
(1)
+ +
x = a bt C t 2 (2) where a-e are constants. The t w for each accelerated condition was calculated from the regression curve obtained. The x - t data observed a t 25 "C were treated by Woolfe's equation16to calculate the tw a t 25 "C and the 95% confidence interval. Figure 1shows the typical activity remaining-time profiles of a-chymotrypsin troche, a-chymotrypsin tablet, bromelain tablets A and B, kallikrein capsule, and 8-galactosidase powder under accelerated degradation conditions. The solid lines in the figure show nonlinear regression curves. Each preparation revealed a different complex profile and no theoretical kinetics could be applied to calculate kinetic parameters. The complexity of the observed profiles may possibly result from the heterogeneity of degradative reactions in the solid state and/or complex kinetics of protein degradation. It is known that heterogeneous degradation of drugs in solid dosage forms often exhibits complex profiles of a similar nature to those observed here. On the other hand, kinetic studies have shown that protein degradation may conform to various kinetic m0dels.~llJ~J7 In the present study bromelain tablets A and B showed different types of degradation curves, since one fit a parabolic curve while the other fit exponential kinetics. The difference in the degradation profile of bromelain between tablets suggests that the complex degradation time courses observed for the preparations studied reflects the heterogeneous solid-state degradation behavior rather than complex protein degradation kinetics. The reciprocal of the time required for 10% inactivation, l/tw, was calculated from the nonlinear regression curve and used as a measure of the inactivation rate.
0022-3549/94/ 1200-454$04.50/0
0 1994, American Chemical Society and American Pharmaceutical Association
4
100
-
80
2
h
-
c
4 I
$
60
M
. -
.'B
g
-2
40
.m 0
c
d
A
.*
c
c)
.?, *
0
v
E * v
-4
4
20
9
t
-6
.A.
0
200
. 400
,
.
'
. 800
600
.
1
.
1000
,
I
I
1200
3.2
3.0
Time ( h r )
Flgure 1-Inacthration of a-chymottypsintroche ( 0 )and tablet ('I at) 60 and 75% RH, bromelain tablets A (A)and B (0)at 50 *Cand 75% RH, kallikrein capsule at 55 O C and 75% RH (O),and 0-galactosidase powder at 60 O C and 50% RH (A). OC
3.0
3.2 1000/T
3.4
(K-l)
Flgure 3-The effect of temperature on inactivation of a-chymottypsin troch (A),a-chymotrypsintablet (A),/%gaabctosidasepowder (O),bromelaln tablet A (V),bromelain tablet B (V),and kallikrein capsule (0).
Table 1-Shelf-Lives
of Commercial Protein Preparations at 25
"c (f00,25)
100
-
3.4
1000/T ( K - l )
confidence interval) apparent Observed estimateda €ab (month) (month) (kcai/moi)
f90,25 (95 % 80
Preparation
2
M
Iga
a-Chymotrypsin troche a-Chymotrypsin tablet Brornelain tablet A Bromeiain tablet B Kallikrein capsule P-Galactosidase powder
60
E
E
A
X .* +
40
8 0
. . . . .I . ........I . . .
I......-...
0 0
100
200
300 Time (hr)
Time
400
8.0 (3.5-12.5) 11.2 (5.7-17.0) 7.1 (2.4-11.7) 4.2 (0.8-7.4) 0.8 (0.2-1.3) 4.3 (2.2-6.5)
5.1 (2.1-12.1) 9.4 (0.5-171) 6.0(2.4-15.0) 1.7 (0.6-5.1) 0.7 (0.6-0.8) 2.9 (0.03-241)
15.6 15.6 11.6 13.5 12.1 17.9
Calculated from the data obtained at accelerated conditions. Calculated from the data obtained at 25 "C and under accelerated degradation conditions. ......I.........,
500
600
(month)
Flgure 2-Inactivation of a-chymotrypsintroche at 50 OC (A), 60 OC (A), and 70 O C (0)(a)and at 25 "C(0)(b). RH was 75 YO. Solid lines represent regression curve and 95% confidence intervals.
Figures 2 shows the remaining activity-time profiles of the a-chymotrypsin troche observed at elevated temperatures ( a ) and that observed at 25 "C (b). The degradation up to 10-20% at 25 "C could be fitted by a linear regression curve. Similar curves were observed for the other preparations. The tw and 95% confidence interval calculated by Woolfe's equation for each preparation are shown in Table 1. Figure 3 shows the Arrhenius plots for the l / t w observed at each temperature. The solid lines were calculated by linearregression analysis. An approximately linear relationship was observed in the temperature range studied for the preparations. The apparent activation energy was calculated from the slope of each line, and these are shown in Table 1.
The tw at 25 "C was estimated from the Arrhenius plots of the data obtained at accelerated degradation conditions above 40 "C for each preparation, and these values and 95 % confidence intervals are shown in Table 1. All the estimates fell within the 95% confidence intervals of the tw calculated from the inactivation data at 25 "C. For some preparations, however, a significant difference was observed between the estimated and observed tw. The bromelain tablet B was the typical case. The tw of this preparation estimated from the accelerated data was much smaller than that observed at 25 "C, indicating that the activation energy in the temperature range above 40 "C tends to be smaller than that in lower temperature range for this preparation. It is often the case for degradation of solid-dosageforms (even for a drug of a small molecular weight) that the Arrhenius plots deviate from a linear relationship. This can be ascribed to the change of a rate-determining step with temperature in heterogeneous reactions (e.g. change from water absorption step to chemical breakdown step). The small degree of deviation observed in the Arrhenius plots for the inactivation of the protein preparations studied may thus be due to the heterogeneity of the solid system rather than to the fit of protein degradation to Arrhenius behavior per se. The t w estimated from the accelerated data of the a-chymotrypsin tablet and 0-galactosidasepowder provided large 95 3'% confidence intervals, since the Arrhenius plots of these accelerated data were not linear. This indicates that accelerated Journal of Pharmaceutical Sciences / 455 Vol. 83, No. 3, March 1994
degradation testing has to be carefully designed regarding the number of temperature levels, etc., in order to get reliable tw estimates. In conclusion, though the inactivation of six Protein PreParations studied showed complex kinetics, the reciprocal of the tw, a measure of inactivation rate, exhibited an approximately linear Arrhenius relationship. The resultsindicate that it may be reasonable in gome cases to extrapolate the results obtained at higher temperature to predict the shelf-life of protein preparations.
References and Notes 1. Gu, K. M.; Erdos, E. A.; Chian , H.; Calderwood, T.; Tsai, K.; Visor, G. C.; Duffy, J.; Hsu, W.; jester, L. C. Pharm. Res. 1991,8, 485-490. 2. Pearlman, R.;Nguyen, T. J. Pharm. Pharmacol. 1992,44(Suppl. l), 178-185. 3. Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7,703-711. 4. Patel, K.; Borchardt, R. T. Pharm. Res. 1990,787-793. 5. Helm, V. J.; Muller, B. W. Pharm. Res. 1990,1253-1256. 6. Motto, M.G.; Hamburg, P. F.; Graden, D. A.; Shaw, C. J.; Cotter, M. L. J. Pharm. Sci. 1991,80,419-423. 7. Friedman, M.; Masters, P. M. J. Food Sci. 1982,47, 760-764. 8. Cole, B.R.; Leadbeater, L. J. Pharm. Pharmacol. 1968,20,48-53.
456 / Journal of Pharmaceutical Sciences Vol. 83. No. 3, March 1994
9. Geigert, J.; Ziegler, D. L.; Panschar, B. M.; Creasey, A. A.; Vitt, C. R. J. Interferon Res. 1987,7, 203-211. 10. Sugiura, M.; Kurobe, M.; Tamura, S.; Ikeda, S. Chem.Pharm.Bull. 1981,29,2096-2100. 11. Patel, J. P.Drug. Dev. Ind. Pharm. 1990,16,2613-2626. 12. U.S. Pharmacopia XXZZ, 1990. 13. Tmimoto, T. GekkanYakuji 1983,259 1183-1188. 14. Yoshioka, s-;IZutSU, K.; h 0 , y.; Takeda, y. Pharm. Res. 1991.8, 480-484. 15. Izutsu, K.;Yoshioka, S.; Takeda, Y. Chem. Pharm. Bull. 199O,s, 800-803. 16. Woolfe, A. J.; Worthington, H. E. C. Drug Dev. Commun. 19741975,1,185-210. 17. Ahern, T.J.; Kilibanov, A. M. Science 1985,228,1280-1284.
SUMIE YOSHIOKA~,YUKIO Aso, KEN-ICHI IZUTSU, AND TADAOTERAO National Institute of Health Sciences 1-18-1, Kamlyoga, Setagaya-ku Tokyo 158, Japan Received November 5, 1992. Accepted for publication December 27, 1993.