ELSEVIER Journal of Controlled Release 40 (1996) 67-76

journal o f controlled

release

Simultaneous skin permeation of dideoxynucleoside-type anti-HIV drugs

Dae-Duk Kim l, Yie W. Chien *

Controlled Drug-Deliver3, Research Center, Rutgers-The State University of New Jersey College of Pharmacy, 4I-D Gordon Road, Piscataway, NJ 08854, USA

Received 8 May 1995; accepted 25 October 1995

Abstract

The effects of vehicle and enhancer on the simultaneous skin permeation of three dideoxynucleoside-type anti-HIV drugs, Zalcitabine (DDC), Didanosine (DDI), and Zidovudine (AZT), were studied using hairless rat skin at 37°C. After the three drugs were saturated in various volume fractions of ethanol/tricaprylin or ethanol/water cosolvent system for 48 h at 37°C, an in vitro skin permeation study was conducted using Valia-Chien permeation cells for 30 h. In both ethanol/tricaprylin and ethanol/water cosolvent systems, the skin permeation rates of DDC, DDI, and AZT increased as the volume fraction of ethanol increased, reached maximum values at 50% (v/v) and 70-80% (v/v) of ethanol, respectively, and then decreased with further increase in ethanol volume fraction. Addition of 5.0% (v/v) of permeation enhancer, i.e., oleic acid (OA), in the ethanol/water (80:20) cosolvent system significantly increased the skin permeation of these drugs with reduced lag time, but not in the ethanol/tricaprylin (50:50) system. Permeation rates of these drugs increased as OA concentration in the ethanol/water (80:20) cosolvent system increased. The skin permeation of ethanol was also investigated to study the enhancing mechanism of vehicle and oleic acid. In the ethanol/water cosolvent system, the skin permeation rate of ethanol also increased as the volume fraction of ethanol increased, reached the maximum value at 70% (v/v) of ethanol, and decreased with further increase in ethanol volume fraction. Moreover, the permeability coefficient of DDC, DDI and AZT showed a good linear relationship with the permeation rate of ethanol up to 70% (v/v) of ethanol volume fraction. Addition of OA in the ethanol/water (80:20) cosolvent system further increased the skin permeation of ethanol, which suggests the mutual skin permeation enhancing-effect of ethanol and OA.

Keywords: Simultaneous transdermal delivery; Acquired immunodeficiency syndrome (AIDS); Human immunodeficiency virus (HIV); Ethanol; Oleic acid

1. Introduct ion

Clinical trials with FDA-approved 2',3'-dide- oxynucleoside analogs, such as 2',3'-dideoxycytidine

* Corresponding author. Present address: University of Washington, Center for Bio-

engineering and Department of Chemical Engineering, Box 351750, Seattle, WA 98195, USA.

(Zalcitabine, DDC), 2' ,3'-dideoxyinosine (Didanos- ine, DDI), and 3'-azido-3'-deoxythymidine (Zidovud- ine, AZT), showed beneficial results in treating pa- tients infected with human immunodeficiency virus (HIV), which is known to cause acquired immunode- ficiency syndrome (AIDS) [1]. However, because of the short biological half-life (about 1 h) and consid- erable 'first-pass' effect of these drugs [2], conven- tional oral or iv routes showed limitation in treating

0168-3659/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0168-3659(95)00172-7

68 D.-D. Kim. Y. W. Chien / Journal of Controlled Release 40 (1996) 67-76

AIDS since they cannot achieve and maintain a constant plasma level within the therapeutic range for a prolonged duration. In addition, several dose- dependent toxic side effects, particularly bone mar- row suppression for AZT [2] and peripheral neuropa- thy for DDC [3] and DDI [4], often require dosage reduction or even cessation of treatment. Therefore, it was proposed that a non-invasive zero-order deliv- ery is desirable to maintain the expected anti-HIV effect and to avoid the strong side effects which may be attributed to an exceeded plasma level of these drugs immediately after iv or oral administration [5].

Transdermal delivery of anti-HIV drugs is a suit- able method to overcome the problems of conven- tional delivery, since it is very helpful in maintaining a suitable plasma concentration through zero-order delivery. Thus, it can enhance the anti-viral activity, and reduce the frequency and severity of side effects by optimizing blood concentration profiles within the therapeutic range for longer duration. Transdermal delivery can also bypass hepatic 'first-pass' elimina- tion, which will improve the bioavailability of drugs [6]. Nevertheless, research articles on the transdermal delivery of anti-HIV drugs are scarce. Several stud- ies have been reported on transdermal delivery of AZT [7-12] and DDI [13]. However, because of the hydrophilicity and high dose requirement of these drugs, they could not reach a sufficient permeation rate to achieve therapeutic efficacy.

In a previous study, we investigated the effects of vehicles and enhancers on the transdermal delivery of each DDC, DDI and AZT alone, and achieved skin permeation rates considerably greater than those reported in the literature [14]. However, the treat- ment of AIDS by a single-drug therapy has limita- tions due to the dose-dependent toxicity and the development of resistance strain. The combination of two or more anti-HIV drugs has been shown to achieve synergistic inhibition of HIV replication in vitro [15-19] and in clinical studies [20-23]. These synergistic combinations may allow the reduction of dose, and thus can reduce the risk of toxicity, while maintaining a good anti-viral effect and reducing the risk of resistance development. Therefore, we report herein the simultaneous skin permeation of three anti-HIV drugs. The effects of vehicles and en- hancers on attaining the maximum skin permeation

rates of these drugs in combination were investi- gated.

2. Materials and methods

2.1. Materials

DDC, DDI, and AZT were kindly supplied by Hoffmann-La Roche (Nutley, NJ), Bristol-Myers Squibb (Wallingford, CT), and Burroughs Wellcome Co. (Research Triangle Park, NC), respectively. Ethanol U.S.P. (200 proof) was obtained from Florida Distillers Co. (Lake Alfred, FL). Tricaprylin (TCP), oleic acid (OA), gentamicin sulfate, and p-chloro- mercuribenzoic acid were purchased from Sigma Chemical Co. (St. Louis, MO). HPLC grade acetoni- trile was purchased from Fisher Scientific (Pitts- burgh, PA). All other chemicals were reagent grade and were used as received.

2.2. Preparation of fuzzy hairless rat skin

Hairless rats (fuzzy strain, 7 -8 weeks), purchased from Harlen Sprague Dawley Inc. (Indianapolis, IN), were killed in a CO 2 chamber on the day of experi- ments. A full-thickness skin with an area approxi- mately 16 c m 2 w a s surgically removed from the dorsal site of each rat. After carefully cleaning the skin with normal saline, the skin specimen was cut into a 4 cm 2 square for experiment, and the thick- ness of each skin was measured by micrometer.

2.3. Preparation of the saturated solutions

Ethanol/TCP and ethanol/water cosolvent sys- tems were chosen as lipophilic and hydrophilic vehi- cles, respectively. Excess amounts of DDC, DDI and AZT, with and without OA, were simultaneously added to the various volume fraction of cosolvent systems (15 ml each) and mixed by vortexing. The solution was immersed in a shaking water bath at 37°C, and allowed to equilibrate for 48 h. To mea- sure the solubility of these drugs in cosolvent sys- tems, approximately 1 ml of saturated solutions were filtered through Teflon filters (0.22 /xm, Micron Separation Inc., Westboro, MO), and the concentra-

D.-D. Kim, Y. W. Chien / Journal of Controlled Release 40 (1996) 67-76 69

tion of drugs was analyzed by HPLC after appropri- ate dilution.

2.4. Hairless rat skin permeation study

Hydrodynamically well-calibrated Valia-Chien permeation systems were used to conduct in vitro hairless rat skin permeation study of DDC, DDI, and AZT at 37°C. Freshly excised hairless rat skin of the dorsal site was mounted on the permeation cells. The donor half-cells, which faced the stratum corneum surface, contained saturated solution of DDC, DDI, and AZT in various compositions of cosolvent sys- tems with or without OA (3.5 ml). The receptor half-cells, which faced the dermis side, were filled with isotonic phosphate buffer (pH 7.4, 3.5 ml) containing 0.01% ( w / v ) gentamicin and 0.01% ( w / v ) p-chloromercuribenzoic acid to minimize the degradation during the permeation study and analysis [24]. At predetermined time intervals, a sample (0.1- 2 ml) was taken from the receptor solution, and refilled with the same volume of fresh receptor solution. Samples were kept in the freezer ( - 2 0 ° C ) until analyzed by HPLC.

2.5. Skin permeation of ethanol

The experimental conditions used for the skin permeation of ethanol was the same as that outlined above for the skin permeation of drugs, except that donor solution was the various compositions of co- solvent systems without drugs. At predetermined time intervals, a sample (100 /xl) was taken from the receptor solution, and refilled with the same volume of fresh receptor solution. Samples were immediately analyzed by GC.

2.6. Analytical methods

DDC, DDI, and AZT concentrations were deter- mined using a gradient reverse-phase HPLC system (HP 1050 Liquid Chromatograph) equipped with a HP 1050 UV detector and a HP 3396A integrator (Hewlett Packard, Mountainview, CA). An HP Hy- persil ODS column (5 /~m, 200 × 4.6 ram, Hewlett Packard) was used as the analytical column main- tained at 37°C. Acetonitrile-phosphate buffer (pH 7.0, 20 mM dibasic sodium phosphate) combination

was used as the mobile phase, which was pro- grammed to change acetonitrile from 5% to 30% over 10 min, and maintain 30% acetonitrile for 5 rain at a constant flow rate of 1.0 ml /min . The injector volume was 15 /xl, and the wavelength was 265 nm. Retention times of DDC, DDI, and AZT were 4.62. 5.21, and 7.16 rain, respectively. The detection sensi- tivity of this HPLC method was 0.2 /xg/ml for DDC, DDI, and AZT, with an injection-to-injection variability of less than 1% for intra-day variation, and less than 3% for inter-day variation.

A GC system (Hewlett Packard model 5890A), equipped with a flame ionization detector, was used to determine the permeation of ethanol through hair- less rat skin. The column used was a 25 m fused silica capillary column (coated with 0.20 /xm car- bowax) with an inside diameter of 0.2 mm (Hewlett Packard). Helium was used as the carrier gas, at a flow rate of 0.7 ml /min , with a column head pres- sure of 16 lb/ inch 2. The carrier and the make-up gas together had a flow rate of 30 ml /min . The flow rate of hydrogen was 30 m l / m i n and that of compressed air, 400 ml /min. The oven temperature was set to have an isothermal temperature of 130°C. The injec- tion port and detector were both set at 200°C. Sam- ples (2 /xl each) were injected into the GC system through an automatic injection system, and the reten- tion time of ethanol was 2.23 min.

2. Z Calculation of permeation parameters

The cumulative amount of each drug permeated per unit area was plotted as a function of time, and the steady-state permeation rate (J~s) and lag time (LT, h) were calculated from the slope and x-inter- cept of the linear portion, respectively. The perme- ability coefficient (Ps, cm/h) , diffusion coefficient (D, cm2/h), and skin/vehicle partition coefficient ( K ) were calculated from the following equations:

h 2

LT = 6D (1)

DK J.,s = ~--Cd = PsCd (2)

where h is the thickness of skin (cm), and C d is the saturation solubility of drug in the vehicle (mg/ml) .

3. Results and discussion

"0 .=. eg

E 8.6 t,,, 4-1

E

E

3.1. Effect of vehicle on skin permeation

The permeation profiles of DDC, DDI, and AZT, saturated in the various volume fractions of ethanol/TCP or ethanol/water cosolvent system, across the hairless rat skin at 37°C are shown in Figs. 1 and 2, respectively. The corresponding values of permeation parameters are summarized in Tables 1 and 2, respectively. The skin permeation rates of these drugs increased as the volume fraction of ethanol increased in both e thanol /TCP and ethanol/water cosolvent systems, reached maximum values at 50% (v /v ) and 70-80% (v /v ) of ethanol, respectively, and then decreased with further in- crease in the volume fraction of ethanol.

Although the solubilities of these drugs in e thano l /TCP are lower than those in the ethanol/water system, the skin permeation rates of these drugs from the ethanol/TCP cosolvent system were higher than those from the ethanol/water sys- tem at the same volume fraction of ethanol (Table 1 and 2). This means that the ethanol/TCP cosolvent system resulted in a higher skin permeability of drugs than the ethanol/water system. Moreover, since the diffusivity of drugs was not significantly different in all formulations, a higher permeability of

these drugs from the ethanol/TCP system is due to greater partitioning of these drugs into the skin dur- ing permeation. The skin/vehicle partition coeffi- cients calculated from Eq. 2 showed that the ethanol/TCP cosolvent system resulted significantly higher values than the ethanol/water system (Table 1 and 2). This suggests that a lipophilic vehicle is useful in increasing the skin permeation of hy- drophilic drugs, such as DDC, DDI and AZT, by enhancing the uptake of these drugs onto the lipophilic stratum corneum, which results in a greater concentration gradient inside the skin.

When lipid vehicles are applied onto the skin, liquid components carrying dissolved drug sub- stances are known to enter the intercellular spaces of the stratum comeum by spreading and by capillary action [25]. Drugs and certain vehicle constituents diffuse into the stratum corneum and deeper skin layers. This results in a decrease in the diffusion resistance of the horny layer by interfering with the molecular packing of the dermal lipids and by dis- turbing their order. Furthermore, the dissolving power of the horny layer lipids may be changed. Therefore lipid solubility is a predominant criterion for the transdermal permeation of substances which entel via the lipid routes. The higher the lipid solubility the steeper the concentration gradient in the horn3 layer can be, especially if suspension preparation~ are applied, and the steeper the gradient, the highe:

(a) (b) 3

6 12 18 24 30

% of EtOH

• 0 O 20

2 • 40 [] 50 @ 60 A 70 T o 8o

o 6 12 18 24 30

Time (hr) Time (hr)

6 12 18 24 30

Time (hr)

15

10

(c)

70 D.-D. Kim, E W. Chien / Journal of Controlled Release 40 (1996) 67-76

Fig. 1. Hairless rat skin permeation profiles of (a) DDC, (b) DDI, and (c) AZT saturated, in combination, in various compositions of tl" ethanol/tricaprylin cosolvent system (n = 3).

D.-D. Kim, Y. W. Chien / Journal of Controlled Release 40 (1996) 67- 76 71

1.5

(a) (b) (c) 2.0 2.0 4.0

1.0

0.5

6 12 18 24 30

T i m e (hr)

1.5

"O

¢D

~., O'J

o

m

E

1.0

0.5

3.0

% of EtOH • 0

20 • 40 [] 50 • 60 A 7O

6 12 18 24 30

2.0

1.0

0.0 0.0 0.0 0 0 0

Time (hr)

6 12 18 24 30

T ime (hr)

Fig. 2. Hairless rat skin permeation profiles of (a) DDC, (b) DDI, and (c) AZT saturated, in combination, in various compositions of the ethanol/water cosolvent system (n = 3).

is the permeation rate. AZT is known to be the most lipophilic among the three drugs [26]. In this re- search, AZT shows the highest solubility in lipophilic

ethanol/TCP cosolvent system, and consequently has the highest permeation rate compared to DDC and DDI (Table 1).

Table 1 Effect of volume fraction of ethanol on the hairless rat skin permeation various volume fraction of ethanol/tricaprylin cosolvent system at 37°C

parameters of DDC, DDI and AZT saturated, in combination, in

Drug EtOH/TCP Solubility Flux Lag time Diffusivity Partition coefficient (%, v /v ) [mg/ml (+SD)] [ /~g/cm 2 per h (_+SD)] [h (_+SD)] [cmZ/h (_+SD)](×103) (_+SD)

DDC 0/100 0.01 (0.00) 3.63 (0.73) 2.57 (0.71) 0.40 (0.10) 55.64 (7.77) 20/80 0.41 (0.03) 38.53 (6.59) 2.02 (0.53) 0.52 (0.14) 14.82 (4,63) 40/60 1.67 (0.10) 90.90 (16.48) 2.76 (0.79) 0.38 (0.10) 11.53 (3.04) 50/50 2.54 (0.16) 109.08 (7.70) 6.07 (1.57) 0.17 (0.04) 20.16 (4.68) 60/40 3.44 (0.19) 88.32 (4.56) 4.76 (1.83) 0.23 (0.07) 9.55 (3.88) 70/30 4.06 (0.25) 43.35 (2.80) 6.77 (0.99) 0.15 (0.02) 5.63 (1.06) 80/20 5.34 (0.04) 31.69 (12.02) 8.22 (0.16) 0.12 (0.00) 3.81 (1.50)

100/0 11.25 (1.23) 1.61 (1.16) 12.23 (0.16) 0.08 (0.00) 0.14 (0.10)

DDI 0 / I00 0.04 (0.01) 1.44 (0.19) 4.68 (1.80) 0.24 (0.12) 14.09 (4.66) 20/80 0.36 (0.02) 18.46 (3.86) 5.70 (0.40) 0.17 (0.01) 22.92 (6.49) 40/60 1.17 (0.07) 42.21 (6.08) 7.92 (0.16) 0.13 (0.00) 22.21 (3.38) 50/50 1.61 (0.10) 61.48 (13.75) 8.04 (0.90) 0.12 (0.01) 24.06 (6.77) 60/40 2.14 (0.13) 31.10 (5.85) 5.44 (1.13) 0.19 (0.04) 2.94 (1.16) 70/30 2.38 (0.16) 21.17 (2.73) 5.27 (0.72) 0.19 (0.03) 3.68 (0.88) 80/20 2.99 (0.23) 10.94 (1.75) 7.92 (0.11) 0.13 (0.00) 2.26 (0.38)

100/0 3.99 (0.39) 0.96 (0.30) 12.86 (0.66) 0.08 (0.00) 0.24 (0.07)

AZT 0/100 1.67 (0.11) 9.71 (0.42) 5.92 (2.11) 0.19 (0.08) 2.65 (0.86) 20/80 20.91 (1.06) 203.09 (3.99) 6.61 (1.10) 0.15 (0,02) 4.99 (0.86) 40/60 57.20 (1.12) 294.52 (9.33) 7.21 (0.39) 0.14 (0.01) 2.89 (0.13) 50/50 69.11 (1.01) 453.58 (53.05) 5.98 (1.63) 0.18 (0.05) 2.99 (0.53) 60/40 84.02 (0.94) 229.43 (29.54) 5.00 (1.27) 0.21 (0.05) 1.04 (0.12) 70/30 83.07 (l.01) 187.88 (18.69) 6.81 (0.93) 0.15 (0.02) 1.21 (0.29) 80/20 5.33 (1.29) 145.35 (16.62) 6.03 (1.43) 0.17 (0.04) 0.81 (0.29)

100/0 103.95 (7.87) 8.42 (1.59) 10.62 (0.34) 0.09 (0.00) 0.07 (0.01)

72 D.-D. Kim, E W. Chien / Journal of Controlled Release 40 (1996) 67-76

3.2. Effect o f oleic acid on skin permeation

The two best cosolvent systems, i.e., e thano l /TCP (50:50) and e thanol /water (80:20), were chosen to study further the skin permeation-enhancing poten- tial of OA. As shown in Fig, 3, the addition of 5.0% ( v / v ) OA in the e thano l /TCP (50:50) cosolvent system could not significantly increase the perme- ation rate of DDC, DDI, and AZT achieved without OA. However, when the same amount of OA was added in the e thanol /water (80:20) cosolvent sys- tem, OA dramatically increased the permeation rate of all three drugs: 0.35 (_+0.10), 0.29 (+0 .08) , and 0.47 (+0 .07 ) m g / c m z per h for DDC, DDI, and AZT, respectively,

It has been reported that OA provides a pathway of diminished resistance for drug transport by dis- rupting the intercellular lipid domain of stratum corneum or coexisting as pools in the ordered stra- tum corneum lipid structure [27]. Moreover, the ex- tent of the effect of OA on lipid perturbation and flux is known to be quantitatively related to the amount of OA incorporated into the stratum corneum

0.6

0.5

0 4

' - ~ 0.3

E ~ ~ m 0.2

0.1

0.0 DDC

j DDI

t I EtOH/TCP (50:50) AZT

K///,I EtOH/TCP (50:50) + 5.O%(v/v) OA I~%~ EtOH/water (80:20)

EtOH/water (80:20) + 5.0%(v/v) OA

Fig. 3. Effect of 5% (v/v) oleic acid on hairless rat skin perme- ation rates of DDC, DDI, and AZT saturated, in combination, in ethanol/tricaprylin (50:50) or ethanol/water (80:20) cosolvent system (n = 3).

bilayer [28]. However, the addition of OA in viscous e thano l /TCP system seems to reduce the thermody- namic activity of OA to distribute from the vehicle to the skin, which resulted in no significant enhanc- ing effect.

Table 2 Effect of volume fraction of ethanol on the hairless rat skin permeation various volume fractions of ethanol/water cosolvent system at 37°C

parameters of DDC, DDI and AZT saturated, in combination, in

Drug EtOH/water Solubility Flux Lag t i m e Diffusivity Partition coefficient (%, v/v) [mg/ml(+SD)] [/xg/cm 2 perh(+ SD)] [h (-t-SD)] [cm2/h (_+SD)](× 103) (±SD)

DDC 0/100 122,11 (5.77) 3,19 (0.37) 9.66 (0.91) 0.10 (0.01) 0.02 (0.00) 20/80 167,63 (12.93) 15.36 (2.03) 8.23 (2.04) 0.13 (0.03) 0.06 (0.01) 40/60 168,75 (13.54) 47.51 (6.47) 11.26 (0.68) 0.09 (0.01) 0.25 (0.03) 50/50 171.99 (10.49) 62.95 (9.24) 9.50 (0.26) 0.10 (0.00) 0.27 (0.04) 60/40 135.09 (2.54) 67.45 (10.46) 11.18 (0.39) 0.09 (0.00) 0.43 (0.07) 70/30 122.23 (6.96) 75.69 (15.15) 9.32 (0.47) 0.11 (0.01) 0.45 (0.09) 80/20 83.53 (8.27) 56.82 (7.50) 9.56 (0.68) 0.10 (0.01) 0.51 (0.10)

DDI 0/100 107.51 (4.50) 1.73 (0.59) 13.49 (0.91) 0.07 (0.01) 0.02 (0.01) 20/80 122.89 (9.11) 10.22 (1.29) 9.92 (0.43) 0.10 (0.00) 0.06 (0.01) 40/60 124.32 (8.36) 23.86 (2.81) 12.21 (0.38) 0.08 (0.00) 0.18 (0.02) 50/50 118.44 (7.26) 30.62 (4.65) 9.59 (0.33) 0.10 (0.00) 0.19 (0.03) 60/40 97.33 (2.39) 31.57 (3,25) 11.71 (0.42) 0.08 (0.00) 0.29 (0.02) 70/30 84.24 (4,91) 45.68 (9,97) 10.43 (0.32) 0.10 (0.00) 0.44 (0.10) 80/20 57.16 (5,21) 39.59 (7.31) 10.64 (1.071) 0.09 (0.01) 0.58 (0.17)

AZT 0/100 65.38 (2.80) 1.99 (0.64) 9.75 (2.54) 0.11 (0.03) 0.02 (0.01) 20/80 86.53 (6.07) 9.32 (1.51) 8.86 (0.25) 0.11 (0.00) 0.07 (0.01) 40/60 148.52 (11.90) 33.09 (5.51) 10.88 (0.68) 0.09 (0.01) 0.19 (0.02) 50/50 175.70 (10.67) 38.03 (5.49) 9.09 (0.12) 0.11 (0.00) 0.15 (0.02) 60/40 223.49 (4.01) 74.08 (9.18) 10.12 (0.44) 0.10 (0.00) 0.26 (0.03) 70/30 225.86 (13.89) 101.94 (25.84) 11.09 (0.82) 0.11 (0.01) 0.32 (0.10) 80/20 223.15 (20.13) 159.23 (46.23) 10.62 (0.34) 0.09 (0.01) 0.62 (0.22)

D.-D. Kim, Y. W. Chien / Journal of Controlled Release 40 (1996) 67-76 73

10

i8 ,*-,Or)

+l 6

g, o

(a) lO

8

6

4

2

o 6 12 18 24 30

T i m e (hr)

(b) (c) lO

%(v/v) of OA__ • 5.0 T O 3.0 | -, 2.0 7 / ' O 1.0 / ~

Z 6 12 18 24 30

T ime (hr)

8

6

4

2

0 6 12 18 24 30

T ime (hr)

Fig. 4. Effect of oleic acid (OA) concentration on hairless rat skin permeation profiles of (a) DDC, (b) DDI, and (c) AZT saturated, in combination, in the ethanol/water (80:20) cosolvent system containing various concentrations of OA (n = 3).

The effect of the concentration of OA on the permeation profiles of DDC, DDI, and AZT, satu- rated in ethanol/water (80:20) cosolvent system, across hairless rat skin is shown in Fig. 4. The permeation rates were calculated from the initial linear portion, and summarized in Table 3 together with the permeability and lag time. As increasing concentration of OA in the cosolvent system, the permeation rates of all three drugs increased consid- erably with reduced lag time. Moreover, it is interest-

ing to observe that the permeation rates of drugs decreased after approximately 12 h when more than 2.0% ( v / v ) of OA was added in the ethanol/water (80:20) cosolvent system (Fig. 4). As reported by Berner et al. [29], drug skin permeation has a linear dependence on the ethanol flux for ethanol volume fractions < 70%. The addition of OA in ethanol/water cosolvent system seems to increase the flux of ethanol, as well as the drugs. Moreover, the depletion of ethanol in the donor solution during

Table 3 Effect of oleic acid (OA) concentration on hairless rat skin permeation parameters of DDC, DDI and AZT saturated, in combination, in ethanol/water (80:20) cosolvent system at 37°C

Drug % of OA Flux Permeability Lag time (%, v/v) [ ~g /cm -~ per b (±SD)] [cmZ/h (_+ SD)] (X 103) (h _+SD)

DDC 0 56.82 (7.49) 0.6802 (00897) 9.57 (0.69) 1.0 225.77 (12.06) 2.7133 (0.1449) 9.50 (0.31) 2.0 270.12 (82.83) 3.2154 (0.9859) 2.20 (0.19) 3.0 336.43 (132.33) 3.9823 (1.5665) 2.39 (0.32) 5.0 346.45 (100.54) 4.1298 (1.1985) 1.38 (0.03)

DDI 0 39.59 (7.31) 0.6927 (0.1278) 10.64 (1.07) 1.0 113.83 (11.10) 1.9640 (0.1914) 10.48 (0.58) 2.0 186.53 (20.39) 3.2154 (0.3515) 2.45 (0.20) 3.0 268.04 (37.13) 4.6647 (0.6462) 2.88 (0.52) 5.0 291.27 (84.42) 5.1579 (1.4950) 2.37 (0.16)

AZT 0 159.23 (46.23) 0.7135 (0.2072) 11.09 (0.82) 1.0 270.90 (18.33) 1.2101 (0.0819) 10.03 (1.00) 2.0 321.90 (34.29) 1.4366 (0.1530) 1.91 (0.44) 3.0 412.29 (50.99) 1.8424 (0.2279) 2.06 (0.29) 5.0 468.46 (66.80) 2.1170 (0.03019) 1.68 (0.54)

74 D.-D. Kim, Y. W. Chien / Journal of Controlled Release 40 (1996) 67- 76

400 10

~ 300

~ ~ 100 E - i

0 0

(a) (b)

%(v/v) of EtOH • 20

40 • 50 T

60 V 70 T J T {3 80 ~ ±

6 12 18 24 30

Time (hr)

100

.H

i 10

8

.E ~. 0.1

0 I I I I I

20 40 60 80 100

%(vlv) of EtOH

Fig. 5. (a) Permeation profiles and (b) permeation rate of ethanol across the hairless rat skin from the various compositions of the ethanol/water cosolvent system (n = 3).

the course of permeation probably resulted in the reduction of the permeability of the drugs. The mu- tual permeation enhancing effect of ethanol and OA was studied by determining skin permeation of ethanol.

3.3. Sk in p e r m e a t i o n o f e thano l

Fig. 5a shows the hairless rat skin permeation profiles of ethanol from various compositions of e thanol /water cosolvent system at 37°C. The perme- ation rate of ethanol increased as the volume fraction of ethanol increased up to < 70% (v /v ) , and then

1.0 • DDC (~=0.938) [] DDI (¢ --0.996)

o~ 0.8 A AZF (r" 0.957~/= " T / .

0,6

w 0.4

0.2 Q

D. 0.0 i t

5 10 15 Permeation rate of ethanol

(mglcm21hr _* SD)

Fig. 6. Linear dependency of skin permeability of DDC, bDl and AZT on the permeation rate of ethanol from the ethanol/water cosolvent system (n = 3).

decreased with further increase in volume fraction of ethanol (Fig. 5b). Moreover, the hairless rat skin permeability of DDC, DDI and AZT from the ethanol /water cosolvent system showed a good lin- ear relationship with the permeation rate of ethanol up to 70% ( v / v ) of ethanol (Fig. 6).

It was suggested that the linear dependence of nitroglycerin skin permeation on ethanol flux for ethanol volume fractions < 70% resulted from the two permeants following each other through the stratum corneum and experiencing the same skin- solvent interactions [29]. Recently, it was also re- ported that the enhanced permeation of estradiol from vehicles with ethanol concentrations up to 60% ( w / w ) is partially related to the increased drug solubility in the stratum corneum, and the decreased estradiol flux from vehicles with higher ethanol con- centrations is due to ethanol dehydration effects on the stratum corneum [30]. The good linear relation- ship between the skin permeability of drug and the permeation rate of ethanol in this study (Fig. 6; implies that ethanol and drug co-permeate througl: the skin during the course of skin permeation experi- ment, and experience the same skin-solvent interac- tions, which enhance the drug permeability by in- creasing drug solubility in stratum corneum [29]. Th( decreased permeation rate of ethanol with ethano concentration higher than 70% ( v / v ) is probably du~ to the dehydration of stratum corneum, which in. creased barrier properties to ethanol permeatior [29,30].

D.-D. Kim, KW. Chien / Journal of Controlled Release 40 (1996) 67-76 75

50

9 ~ 40

wo T

~ ao

~ 10 I:L

0 I I I I I 0 1 2 3 4 5

%(v/v) of oleic acid

Fig. 7. Effect of oleic acid (OA) concentration on the hairless rat skin permeation rate of ethanol from various compositions of the ethanol/water cosolvent system containing various concentrations of OA (n = 3).

drugs , w h i c h can ach i eve a synerg i s t i c a n t i - H I V

ac t iv i ty wi th r educed toxici ty .

Acknowledgements

The au thors wou ld l ike to express the i r apprec ia -

t ion to H o f f m a n n - L a Roche , B r i s t o l - M y e r s Squ ibb ,

and B u r r o u g h s W e l l c o m e Co. for the i r d o n a t i o n o f

D D C , D D I and A Z T , respec t ive ly . Th i s r e s e a r c h was

par t ia l ly suppo r t ed by J o h n s o n and J o h n s o n Predoc-

toral R e s e a r c h F e l l o w s h i p a w a r d e d to D.-D.K.

References

Moreover, Fig. 7 shows that the skin permeation rate of ethanol further increases with the addition of OA in ethanol/water (80:20) cosolvent system. OA seems to enhance the skin permeation of ethanol by diminishing the skin barrier property to permeation, which further enhanced the skin permeation of drugs. Therefore, it can be speculated that the addition of OA in ethanol/water cosolvent system enhances the skin permeation of drugs (Fig. 4 and Table 3) by the mutual permeation enhancing effect of ethanol and OA.

4. Conclusion

Ethanol/TCP and ethanol/water cosolvent sys- tems enhanced skin permeation of DDC, DDI and AZT combination by different mechanism(s). Addi- tion of OA in ethanol/water (80:20) dramatically enhanced skin permeation of these drugs, but not in the ethanol/TCP (50:50) cosolvent system. En- hanced skin permeability of drugs achieved by ethanol/water cosolvent system for up to 70% (v/v) ethanol had a linear relationship with the skin perme- ation rate of ethanol. Moreover, OA incorporated in the ethanol/water (80:20) cosolvent system further e n h a n c e d the skin p e r m e a t i o n o f e thanol , i m p l y i n g a

m u t u a l e n h a n c i n g ef fec t o f e t hano l and OA. T h e s e

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d e r m a l de l ive ry o f d i d e o x y n u c l e o s i d e - t y p e a n t i - H I V

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