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Biomembrane Permeation and Stereochemistry
COMMUNICATI0NS
Biomembrane Permeation and Stereochemistry To the Editor: Over the years, we have investigated the transdermal dual-controlled systemic delivery of levonorgestrel, the potent l-stereoisomer of synthetic progestin dl-norgestrel, and of estradiol-l7P, a biologically active p-stereoisomer of estradiol, from a microreservoir-type transdermal drug delivery system.1This microreservoir-type transdermal drug delivery system was evaluated clinically in 12 females of child-bearing age. By transdermal dual-controlled systemic delivery, this combination of levonorgestrel and estradiol-17P was found effective in suppressing the cyclic surge of progesterone and thus achieved the objective of contraception. The skin permeation kinetics of dl- and I-norgestrel (levonorgestrel) was recently studied with human cadaver skin.2 The results indicated that the permeation rates of dl-norgestrel across the intact skin (excised from six regions on the same human cadaver) are significantly higher than those of 1-norgestrel (p < 0.01). Apparently, the stereochemical configuration plays a role in the skin permeation of norgestrel. We propose that the effect of stereochemistry could also occur in the permeation of stereoisomers across other biological membranes. To provide experimental evidence, we have investigated the biomembrane permeation of three pairs of steroid-type stereoisomers in addition to dl- and 1-norgestrel. These pairs of stereoisomers have a steroidal skeleton similar to that of norgestrel, but they have a-and P-OH groups positioned at different locations on the steroidal skeleton. To confirm the important role of stereochemistry, we also performed permeation kinetics studies of dl- and l-norgestrel across ear pinna skin and various absorptive mucosa specimens, which were freshly excised from the same rabbits and mounted in the Valia-Chien permeation cell3 for 24 (mucosa) or 72 h (skin). The permeation across human cadaver skin was also studied for comparison. Each cell consisted of a donor half-cell, which contained the saturated solution of dl- or I-norgestrel in a simulated physiological fluid, and a receptor half-cell, which contained an isotonic phosphate buffer (at pH 7.4). All cells were maintained a t body temperature by circulating 37 "C water through the water jacket surrounding the solution compartment of each half-cell. Aliquots (25 p L each) were taken at predetermined time intervals throughout the course of permeation studies and analyzed for dl- or 1-norgestrel concentration in the samples by highperformance liquid chromatography. The permeation profiles of dl- and 1-norgestrel across the skin excised from a female human cadaver (Figure 1)indicate that both dZ- and 1-norgestrel permeate through the human cadaver skin according to zero-order kinetics. The same constant permeation profiles were also obtained for permeation through all the biomembranes studied. The results (Table I) suggest that the permeation rates of dl- and I-norgestrel vary from one biomembrane to another. Similar to the skin permeation rate observed, the permeation rates of dl-norgestrel across various mucosae are consistently higher than those for I-norgestrel. Because d- and 1-norgestrel cannot be easily separated chromatographically, the concentration of dl-norgestrel in 342 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
6.0 -
Table I-Effect of Stereochemical Conflguration of Norgestrel on Permeation Rate across Biomembranes
Region Human Skin Male cadaverC Chest Abdomen Arm Hip
Back Thigh
Female cadavere Back Rabbit' Skin (ear pinna) Buccal mucosa Nasal mucosa Rectal mucosa Vaginal mucosa
Permeation Rate, ng/cm2/ha d/-Norgestrel kNorgestre1 d-NorgestrelD
74.3 65.5 51.0 58.6 39.3 35.3
(l.l)d (4.1) (7.6) (1.1) (6.2) (6.3)
78.8 (14.5) 51.2 (7.3) 287.0 (68.6) 1780.0 (282.5) 590.3 (155.4) 571 .O (1 23.2)
47.8 35.9 34.5 28.7 26.6 25.3
(7.3) (4.9) (6.1) (4.3) (3.4) (3.6)
26.5 29.6 16.5 29.9 12.7 10.0
44.8
(2.8)
34.0
41.0 (7.1) 208.3 (38.6) 861 .O (1 19.0) 322.3 (65.2) 396.3 (39.4)
10.2 78.7 919.0 268.0 174.7
a The skin permeation rates for dk and knorgestrel were from ref 2. Calculated from eq 5. For each skin region, n = 5 (excised from the same adult male cadaver). Numbers in parentheses are standard deviations (SD). n = 5 (excisedfrom a 34-year old female cadaver). n = 5 for skin region and n = 3 for all mucosal regions (excised from the same rabbits).
the receptor solution, determined by high-performance liquid chromatography, could be considered as the sum of d- and 1-norgestrel. Then, the cumulative amount of dl-norgestrel permeating through a skin specimen may be described by:
c,
In eq 1,P,is the skin permeability of norgestrel, c d and are the concentrations of norgestrel in the donor and receptor solutions, respectively, and t is the duration of a permeation study. The subscripts (d) and (1) denote, respectively, the d and 1-stereoisomers of norgestrel. If the conditions C,,,, >> CrCd)and c d ( ( ) >> C,,,, are 0022-3549/93/0300-0342$02.50/0 0 1993, American Pharmaceutical Association
150
-x
- B U C W MUCOU
800
1::
120.
4 80.
(3)
NAUL MUCOSA
Equation 3 is valid because:
240 120
Substituting eqs 4a and 4b into eq 3 results in eq 5: 0
5
10
15
20
21
0
15 TIME (hn)
5
llYE (hn)
250
1
RECTAL HUCOU
1
'21
20
10
25
[ VAGINAL HUCOSA
q 200
Therefore the skin permeation rate of d-norgestrel can be calculated, using eq 5, from the experimental values of (Q/t),, and (Q/t)l. The results in Table I also suggest that the permeation rates of I-norgestrel across various biomembranes are mostly greater than those of d-norgestrel. To confirm the effect of stereochemistry on biomembrane permeation observed for norgestrel (Table I), we further investigated the permeation kinetics of the a-and p-stereoisomers of hydroxyprogesterone (11-and 20-OH) and estradiol (17-OH) across the skin and various absorptive mucosae from rabbit. Again,a zero-order kinetics was observed for the permeation of these pairs of stereoisomers across all the biomembranes studied (Figure 2). Similar to d- and l-norgestrel, these a-and p-stereoisomers show dependence on stereochemical configuration, with the a-isomer yielding a higher permeation profile than the p-isomer. To compare the effect of stereochemistry on the permeation through these biomembranes with different thicknesses, the following equation was applied to correct the difference in membrane thickness:
4 150
0
5
10
15
20
25
0
5
10
llME (hn)
20
15
25
TIME (hn)
Figure 2-Comparative permeation profiles of (0)a-and (0)p-stereoisomers of 11-hydroxyprogesterone across rabbit skin and absorptive mucosae. Data shown are mean values ( A one SD) of triplicate determinations.
In eq 6, (Qlt), and (Qlt), are, respectively, the normalized and apparent rates of permeation across the biomembrane with a thickness of h,. The results (Table 11) indicate that the normalized permeation rate is dependent on the stereochemi d configuration. The degree of dependence varies from one type of stereoisomer to another, the position of stereoactive group, and the nature of biomembrane. The effect of donor concentration on the rate of transmucosal permeation was also investigated with one pair of stereoisomers. The results (Table 111) indicate that the normalized permeation rates obtained with diluted solution
maintained throughout the course of skin permeation study, then eq 1can be simplified to:
Thus, the rate of permeation is described by:
Table 11-Effect of Stereochemlcal Conflguratlon on the Normallzed Permeation Rate of Steroids from Saturated Solution'
Thickness, cmc
Biomembraneb
Mucosa Buccal Nasal Rectal Vaginal Skin Ear pinna
0.10 0.05
0.125 0.15 0.05
Normalized Permeation Rate, pglcmlh' Hydroxyprogesteronee
NorgestreP
Estradiol-17 0 H e
20-OH
11-OH
d
I
a
P
ff
P
a
B
0.008 0.046 0.036
0.021
0.618 1.066
0.209
0.043 0.040
0.056 0.205 0.140 0.183
0.074 0.181 0.167 0.298
0.040 0.228
1.053 0.710
0.703 0.520 0.346
0.080 0.195 0.160
0.026
0.059
0.001
0.002
0.01 1
0.006
0.016
0.018
0.005
0.163 0.146
0.233 0.005
a Donor solution contained excess amount of the drug to maintain saturated concentration.* Freshly excised from the same rabbits (n = 3). Average of six determinations. Calculated from normalized permeation rate = permeation rate x membrane thickness. Variability values in the permeation rate data represented by the coefficient of variation are: norgestrel, 14.3-26.4% (d/)and 10.0-20.3% (Cisomer);11-OH-progesterone,2230.3% (aisomer)and 2.9-43.4% (p-isomer);20-0H-progesterone,4448.4% (a-isomer)and 6.034.9%(p-isomer);estradiol-l7-OH,2.3-1 9.4% (a-isomer) and 6.4-34.4% (p-isomer).
'
Journal of Pharmaceutical Sciences I 343 Vol. 82, No. 3, March 1993
Table lll-comparlson of the Predicted and Obsenred Normallzed Permeation Rates of 11-Hydroxyprogesterone from Diluted Solutiona Normalized Permeation Rate, pg/cm/hc Mucosab
a-Isomer Predictedd ~~
Buccal Nasal Rectal Vaginal
p-Isomer
Observede
Predicted"
Observed"
~
0.124 (0.003) 0.140(0.017) 0.042 (0.002) 0.059(0.006) 0.213(0.038) 0.220(0.028) 0.141 (0.086) 0.086(0.008) 0.211 (0.064) 0.232 (0.030) 0.104(0.015) 0.096(0.008) 0.142(0.016) 0.263(0.061) 0.069 (0.030) 0.070(0.005)
'Donor solution has drug concentration at 20% of the saturated concentration of the drug. Freshly excised from the same rabbits (n = 3). Calculated from normalized permeation rate = permeation rate x membrane thickness (numbers in parentheses are SDs). Calculated from the data in Table II by correcting the fivefold difference in donor drug concentration. Variabilities in the permeation rate data represented by the coefficients of variation: 12.1-23.2% (aisomer) and 7.1-10.2% (pisomer). follow v e r y m u c h t h e same t r e n d as those observed w i t h saturated solution. With only one exception, all t h e ob-
344 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
served d a t a a r e in good agreement w i t h t h e predicted values. The agreement suggests that t h e stereochemical configurations g i v e r i s e t o t h e difference in permeation rates obtained.
References and Notes 1. Chien, Y.W.; Chien, T. Y.;Bagdon, R. E.; Huang,Y. C.; Bierman, R. H . Pharm. Res. 1989, 6, 1000-1010. 2. Chen. G.-S.: Gone. S.-J.: Zhou. R.-R: Xie. X . 4 . Acta Pharmaeol. Sin& 1991',12,Z37-446. 3. Chien, Y. W.; Valia, K. H. Drug Dev. Ind. Pharm. 1984, 10, 575-599. I
,
YIE W. C H ~ E N ~
MONANAIR Controlled Drug-Delivery Research Center Rutgers University, College of Pharmacy Piscataway, NJ 08855-0789
Received February 20, 1992. Accepted for publication October 5, 1992.
Biomembrane Permeation and Stereochemistry To the Editor: Over the years, we have investigated the transdermal dual-controlled systemic delivery of levonorgestrel, the potent l-stereoisomer of synthetic progestin dl-norgestrel, and of estradiol-l7P, a biologically active p-stereoisomer of estradiol, from a microreservoir-type transdermal drug delivery system.1This microreservoir-type transdermal drug delivery system was evaluated clinically in 12 females of child-bearing age. By transdermal dual-controlled systemic delivery, this combination of levonorgestrel and estradiol-17P was found effective in suppressing the cyclic surge of progesterone and thus achieved the objective of contraception. The skin permeation kinetics of dl- and I-norgestrel (levonorgestrel) was recently studied with human cadaver skin.2 The results indicated that the permeation rates of dl-norgestrel across the intact skin (excised from six regions on the same human cadaver) are significantly higher than those of 1-norgestrel (p < 0.01). Apparently, the stereochemical configuration plays a role in the skin permeation of norgestrel. We propose that the effect of stereochemistry could also occur in the permeation of stereoisomers across other biological membranes. To provide experimental evidence, we have investigated the biomembrane permeation of three pairs of steroid-type stereoisomers in addition to dl- and 1-norgestrel. These pairs of stereoisomers have a steroidal skeleton similar to that of norgestrel, but they have a-and P-OH groups positioned at different locations on the steroidal skeleton. To confirm the important role of stereochemistry, we also performed permeation kinetics studies of dl- and l-norgestrel across ear pinna skin and various absorptive mucosa specimens, which were freshly excised from the same rabbits and mounted in the Valia-Chien permeation cell3 for 24 (mucosa) or 72 h (skin). The permeation across human cadaver skin was also studied for comparison. Each cell consisted of a donor half-cell, which contained the saturated solution of dl- or I-norgestrel in a simulated physiological fluid, and a receptor half-cell, which contained an isotonic phosphate buffer (at pH 7.4). All cells were maintained a t body temperature by circulating 37 "C water through the water jacket surrounding the solution compartment of each half-cell. Aliquots (25 p L each) were taken at predetermined time intervals throughout the course of permeation studies and analyzed for dl- or 1-norgestrel concentration in the samples by highperformance liquid chromatography. The permeation profiles of dl- and 1-norgestrel across the skin excised from a female human cadaver (Figure 1)indicate that both dZ- and 1-norgestrel permeate through the human cadaver skin according to zero-order kinetics. The same constant permeation profiles were also obtained for permeation through all the biomembranes studied. The results (Table I) suggest that the permeation rates of dl- and I-norgestrel vary from one biomembrane to another. Similar to the skin permeation rate observed, the permeation rates of dl-norgestrel across various mucosae are consistently higher than those for I-norgestrel. Because d- and 1-norgestrel cannot be easily separated chromatographically, the concentration of dl-norgestrel in 342 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
6.0 -
Table I-Effect of Stereochemical Conflguration of Norgestrel on Permeation Rate across Biomembranes
Region Human Skin Male cadaverC Chest Abdomen Arm Hip
Back Thigh
Female cadavere Back Rabbit' Skin (ear pinna) Buccal mucosa Nasal mucosa Rectal mucosa Vaginal mucosa
Permeation Rate, ng/cm2/ha d/-Norgestrel kNorgestre1 d-NorgestrelD
74.3 65.5 51.0 58.6 39.3 35.3
(l.l)d (4.1) (7.6) (1.1) (6.2) (6.3)
78.8 (14.5) 51.2 (7.3) 287.0 (68.6) 1780.0 (282.5) 590.3 (155.4) 571 .O (1 23.2)
47.8 35.9 34.5 28.7 26.6 25.3
(7.3) (4.9) (6.1) (4.3) (3.4) (3.6)
26.5 29.6 16.5 29.9 12.7 10.0
44.8
(2.8)
34.0
41.0 (7.1) 208.3 (38.6) 861 .O (1 19.0) 322.3 (65.2) 396.3 (39.4)
10.2 78.7 919.0 268.0 174.7
a The skin permeation rates for dk and knorgestrel were from ref 2. Calculated from eq 5. For each skin region, n = 5 (excised from the same adult male cadaver). Numbers in parentheses are standard deviations (SD). n = 5 (excisedfrom a 34-year old female cadaver). n = 5 for skin region and n = 3 for all mucosal regions (excised from the same rabbits).
the receptor solution, determined by high-performance liquid chromatography, could be considered as the sum of d- and 1-norgestrel. Then, the cumulative amount of dl-norgestrel permeating through a skin specimen may be described by:
c,
In eq 1,P,is the skin permeability of norgestrel, c d and are the concentrations of norgestrel in the donor and receptor solutions, respectively, and t is the duration of a permeation study. The subscripts (d) and (1) denote, respectively, the d and 1-stereoisomers of norgestrel. If the conditions C,,,, >> CrCd)and c d ( ( ) >> C,,,, are 0022-3549/93/0300-0342$02.50/0 0 1993, American Pharmaceutical Association
150
-x
- B U C W MUCOU
800
1::
120.
4 80.
(3)
NAUL MUCOSA
Equation 3 is valid because:
240 120
Substituting eqs 4a and 4b into eq 3 results in eq 5: 0
5
10
15
20
21
0
15 TIME (hn)
5
llYE (hn)
250
1
RECTAL HUCOU
1
'21
20
10
25
[ VAGINAL HUCOSA
q 200
Therefore the skin permeation rate of d-norgestrel can be calculated, using eq 5, from the experimental values of (Q/t),, and (Q/t)l. The results in Table I also suggest that the permeation rates of I-norgestrel across various biomembranes are mostly greater than those of d-norgestrel. To confirm the effect of stereochemistry on biomembrane permeation observed for norgestrel (Table I), we further investigated the permeation kinetics of the a-and p-stereoisomers of hydroxyprogesterone (11-and 20-OH) and estradiol (17-OH) across the skin and various absorptive mucosae from rabbit. Again,a zero-order kinetics was observed for the permeation of these pairs of stereoisomers across all the biomembranes studied (Figure 2). Similar to d- and l-norgestrel, these a-and p-stereoisomers show dependence on stereochemical configuration, with the a-isomer yielding a higher permeation profile than the p-isomer. To compare the effect of stereochemistry on the permeation through these biomembranes with different thicknesses, the following equation was applied to correct the difference in membrane thickness:
4 150
0
5
10
15
20
25
0
5
10
llME (hn)
20
15
25
TIME (hn)
Figure 2-Comparative permeation profiles of (0)a-and (0)p-stereoisomers of 11-hydroxyprogesterone across rabbit skin and absorptive mucosae. Data shown are mean values ( A one SD) of triplicate determinations.
In eq 6, (Qlt), and (Qlt), are, respectively, the normalized and apparent rates of permeation across the biomembrane with a thickness of h,. The results (Table 11) indicate that the normalized permeation rate is dependent on the stereochemi d configuration. The degree of dependence varies from one type of stereoisomer to another, the position of stereoactive group, and the nature of biomembrane. The effect of donor concentration on the rate of transmucosal permeation was also investigated with one pair of stereoisomers. The results (Table 111) indicate that the normalized permeation rates obtained with diluted solution
maintained throughout the course of skin permeation study, then eq 1can be simplified to:
Thus, the rate of permeation is described by:
Table 11-Effect of Stereochemlcal Conflguratlon on the Normallzed Permeation Rate of Steroids from Saturated Solution'
Thickness, cmc
Biomembraneb
Mucosa Buccal Nasal Rectal Vaginal Skin Ear pinna
0.10 0.05
0.125 0.15 0.05
Normalized Permeation Rate, pglcmlh' Hydroxyprogesteronee
NorgestreP
Estradiol-17 0 H e
20-OH
11-OH
d
I
a
P
ff
P
a
B
0.008 0.046 0.036
0.021
0.618 1.066
0.209
0.043 0.040
0.056 0.205 0.140 0.183
0.074 0.181 0.167 0.298
0.040 0.228
1.053 0.710
0.703 0.520 0.346
0.080 0.195 0.160
0.026
0.059
0.001
0.002
0.01 1
0.006
0.016
0.018
0.005
0.163 0.146
0.233 0.005
a Donor solution contained excess amount of the drug to maintain saturated concentration.* Freshly excised from the same rabbits (n = 3). Average of six determinations. Calculated from normalized permeation rate = permeation rate x membrane thickness. Variability values in the permeation rate data represented by the coefficient of variation are: norgestrel, 14.3-26.4% (d/)and 10.0-20.3% (Cisomer);11-OH-progesterone,2230.3% (aisomer)and 2.9-43.4% (p-isomer);20-0H-progesterone,4448.4% (a-isomer)and 6.034.9%(p-isomer);estradiol-l7-OH,2.3-1 9.4% (a-isomer) and 6.4-34.4% (p-isomer).
'
Journal of Pharmaceutical Sciences I 343 Vol. 82, No. 3, March 1993
Table lll-comparlson of the Predicted and Obsenred Normallzed Permeation Rates of 11-Hydroxyprogesterone from Diluted Solutiona Normalized Permeation Rate, pg/cm/hc Mucosab
a-Isomer Predictedd ~~
Buccal Nasal Rectal Vaginal
p-Isomer
Observede
Predicted"
Observed"
~
0.124 (0.003) 0.140(0.017) 0.042 (0.002) 0.059(0.006) 0.213(0.038) 0.220(0.028) 0.141 (0.086) 0.086(0.008) 0.211 (0.064) 0.232 (0.030) 0.104(0.015) 0.096(0.008) 0.142(0.016) 0.263(0.061) 0.069 (0.030) 0.070(0.005)
'Donor solution has drug concentration at 20% of the saturated concentration of the drug. Freshly excised from the same rabbits (n = 3). Calculated from normalized permeation rate = permeation rate x membrane thickness (numbers in parentheses are SDs). Calculated from the data in Table II by correcting the fivefold difference in donor drug concentration. Variabilities in the permeation rate data represented by the coefficients of variation: 12.1-23.2% (aisomer) and 7.1-10.2% (pisomer). follow v e r y m u c h t h e same t r e n d as those observed w i t h saturated solution. With only one exception, all t h e ob-
344 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
served d a t a a r e in good agreement w i t h t h e predicted values. The agreement suggests that t h e stereochemical configurations g i v e r i s e t o t h e difference in permeation rates obtained.
References and Notes 1. Chien, Y.W.; Chien, T. Y.;Bagdon, R. E.; Huang,Y. C.; Bierman, R. H . Pharm. Res. 1989, 6, 1000-1010. 2. Chen. G.-S.: Gone. S.-J.: Zhou. R.-R: Xie. X . 4 . Acta Pharmaeol. Sin& 1991',12,Z37-446. 3. Chien, Y. W.; Valia, K. H. Drug Dev. Ind. Pharm. 1984, 10, 575-599. I
,
YIE W. C H ~ E N ~
MONANAIR Controlled Drug-Delivery Research Center Rutgers University, College of Pharmacy Piscataway, NJ 08855-0789
Received February 20, 1992. Accepted for publication October 5, 1992.