Journal of Controlled Release, 7 (1988) 243-250 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

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TRANSDERMAL DELIVERY OF LEVONOR~ESTREL I: ALKANOLS AS PERMEATION ENHANCERS IN VITRO

David Friend, Paul Catz, Jorge Heller Controlled Retease and Biomedical Polymers Program, SRI International, Menlo Park, CA 94025 (U.S. A.)

Jean Reid and Richard Baker Membrane Technology and Research, Men/o Park, CA 94025 (U.S. A.)

(Received November 3, 1987; accepted in revised form March 2, 1988)

The effect of alcoholic penetration enhancers on the transdermal delivery of levonorgestrel CLN) was investigated. A series of alkanols was tested in vitro for their ability to enhance the delivery of LN through excised rat skins. The steady-state flux of LN was measured using donor phase compositions of water/ethanol mixtures, and the neat straight chain alkanols ethanol, propanol, butano~, pentano~~ hexanol, and octanol. The steady-state flux of LN was found to increase as the alkyd chain length increased from C, to C,. This was followed by a decrease in the steady-state flux as the alkyl chain length increased above 1 -butanol. Three secondary alkanols, 2-propanol, 2-butanol, and 2-pentanol were also tested as penetration enhancers. The same trend was evident: steady-state flux was highest for 2-butanol with 2-propanol and 2-pentanol giving lower steady- state fluxes. The flux of LN was lower for secondary alkanols relative to the &orrespond~n~prima~ ulkanol. Propylene glycol was also tested as an enhancer. overall, the steady-state flux using thus penetration enhancer was about that of water, which was low relative to all the alkanols. Flux of LN through human cadaver skin from pure ethanol was about four times lower than through rat skin. A mechanism for permeability enhancement based on the alkanol structure and water- solubility is presented as well as a discussion of the data as it relates to development of a trans- dermal delivery system for LN.

INTRODUCTION

Transdermal drug delivery offers many ad- vantages over conventional routes of drug administration. Elimination of hepatic first pass effects, reduced side-effects through optimiza- tion of the blood concentration-time profile, extended duration of activity, and improved patient compliance are some of the advantages of transdermal medication [ 11. However, the excellent barrier properties of the skin [2,3] eliminate all but the more potent drugs from consideration in transdermal drug delivery sys- tems. Another potential problem is skin irrita- tion, due to the drug, adhesive, or excipients in

the delivery system.

LEUONORGESTAEI.

Levonorgestrel (LN) is an extremely potent contraceptive steroid. It is capable of suppress- ing ovulation at a delivery rate as low as 20 ,ug per day from implants [4]. Thus, its delivery- via the transdermal route is suggested. Even at this low dose requirement, LN alone is not suf- ficiently permeable to meet the daily target de- livery rate. Therefore, we investigated the use of penetration enhancers to increase the trans- dermal flux of LN. Ethanol is used in a trans-

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dermal delivery system for 17P-estradiol, a steroid related structurally to LN [ 5,6]. Ethanol is co-released with the drug to the skin from the device leading to increased permeability of es- tradiol. Based on this, we investigated the abil- ity of ethanol, as well as a variety of other alkanols, to increase the transdermal flux of LN from solutions containing excess solid LN.

The relationship between enhancer struc- ture and penetration enhancement are dis- cussed as well as the relevance of the data to the development of a transdermal delivery sys- tem for LN.

MATERIALS AND METHODS

Materials

Levonorgestrel was a gift from the World Health Organization. The alkanols and propyl- ene glycol (reagent grade ) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. The rats (male Wistar strain) were obtained from Si- monsen Labs, Gilroy, CA. Human cadaver skin was obtained from Stanford University Medi- cal Center. The cadaver (male) was refriger- ated at death; skin excision was performed within 24 h post-mortum.

Methods

Permeability studies with rat skins A system employing up to nine glass Franz

diffusion cells was used for the permeability ex- periments with rat skins. The Franz cells were modified with inlet and outlet receiver phase ports to allow continuous flow through the cells.

The rats (180-220 g) were killed in a CO, chamber, and an approximately 6 cm2 area of full-thickness skin was excised from the shaved abdominal site. After removal of the subcuta- neous fat, the skins were washed with physio- logical saline and used in the permeability experiment within 1 h. The skin was mounted

and clamped between the cell body and the cell cap with the furry side facing upward (donor side). The surface area exposed to the donor phase was 5.07 cm2. The donor phase (ca. 10 ml) was prepared by suspending excess solid LN in the appropriate solvent. The donor phase suspension was applied directly on the skin through the cell cap, which was then sealed with a glass stopper. The receptor phase, in contact with the underside of the skin, was isotonic sa- line at 37°C with 0.1% sodium azide added to prevent bacterial growth. The cells were main- tained at 37°C by thermostatically controlled water which was circulated through a jacket surrounding the cell body. The donor phase temperature was measured at 32°C.

Receiver phase solution was pumped through the diffusion cells by means of a Manostat Cas- sette Pump drive unit. A fraction collector was used to collect the cell effluent. The flow rate was set so that the drug concentration in the receptor phase remained below 10% of satura- tion (0.1 pg/ml). In a typical experiment, the flow rate was 10 ml/h and fractions were col- lected every two hours. Uniform mixing of the drug in the receiver phase was achieved by a small magnetic stirring bar driven by an exter- nal 600 rpm motor.

The silastic tubing used was found to absorb LN and subsequently release it when solutions free of LN were passed through it. To avoid this problem, the tubing was changed frequently. When the tubing was reused, the amount of LN released was measured prior to the experiment and a correction applied to the amount of LN collected.

Permeability study with human cadaver skin

The skin was removed from the thigh of the cadaver with a dermatome (10 x 15-cm2 piece). The skin was wrapped in aluminum foil and stored at 4” C until use (approximately one week). The skins were mounted in teflon flow- through cells, similar to the Franz diffusion cells although with a smaller active surface area (0.32

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cm* ) . LN was suspended in pure ethanol to ex- cess and the resulting suspension placed in the donor chamber. The cells were maintained at 30’ C. The receptor phase (distilled water) was passed below the skin (donor side) at a rate of 1.7 to 2.6 ml/h. Samples were collected every 5 h for 50 h.

Chromatographic analysis The receptor phase from the permeability ex-

periments was analyzed for LN using high- pressure liquid chromatography (HPLC). No sample pretreatment was required. The HPLC analysis was performed on a Waters 840 system consisting of two Model 510 pumps, a Model 481 UV detector, a Model 710B WISP (sample processor), and a Digital Computer Model 350 microprocessor/programmer. The column used was a 4.6 mmx 25 cm, de= 10 ,um, Whatman ODS-3 Partisil C-18. A flow rate of 1.8 ml/min was used, with absorbance monitoring at 243 nm. The mobile phase was acetonitrile/H,O (55/45; v/v); the retention time of LN was 4.5 min.

Solubility of LN in alkanols A known amount of LN was added to 2.0 ml

of the appropriate enhancer at 32°C until sat- uration was reached. A known amount of ad- ditional solvent was used to bring the solubility of LN back to the point of saturation. The point of saturation was then calculated from the amount of LN and the amount of solvent.

Octanol/water partition coefficient of LN The octanol/water partition coefficient for

LN was determined at 24’ C. The aqueous phase was buffered to pH 7.4 with 0.05 M sodium phosphate. Equal portions (5 ml) of 1-octanol and buffer were used. LN was dissolved in oc- tanol at 0.13 mg/ml and the two phases were then mixed by inversion in a screw-top test tube for 5 min. After centrifuging (500 g, 5 min) to ensure separation of the phases, the aqueous phase was analyzed for LN using HPLC. The

concentration of LN in the octanol phase was determined by difference.

RESULTS

Permeation of LN through rat skin in vitro

The target delivery rate of LN in women is 25 pg/day (about 1.0 ,ug/h). Based on a 10 cm2 device, the flux required to deliver the target dose of LN is 0.1 ,ug/ (cm” h). The in. vitro ex- periments were performed with rat skin, which is generally more permeable than human skin [7,8]. A preliminary experiment with human cadaver skin (see below) indicated that the flux of LN was about four times lower through hu- man skin relative to rat skin. Therefore, the target flux of LN through rat skin is 0.4 ,ug/ (cm’h).

To meet this target, a permeation enhancer is required, as LN alone is insufficiently perme- able through skin to deliver 25 ,ug/day with an acceptable patch size. Ethanol has been shown to increase the permeability of 17/3-estradiol through human skin [ 5,6]. We therefore inves- tigated the use of ethanol as a permeation en- hancer for LN. Release profiles (flux against time) are shown in Figs. 1 and 2 for enhancer compositions of water/ethanol. As can be seen in Fig. 1, the steady-state flux of LN through

,- r 0.06

“$ r 7 0.04

?z 20% EtOH 2 0 02 z A

0.00 0 20 40 6b sb

TIME (h)

Fig. 1. FluxofLN throughrat skin in uitrousingO% (rz=a), 20% (n=4), and 40% (n=3) ethanol in water as donor phase solvent. Error bars indicate mean standard errors.

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80% EtOH - 0.08 \ r “E

0 0 06 T 7

- 0.04 X

3 LL 0.02

f

0.00

0 20 40 60 60

TIME(h)

Fig. 2. Flux of LN through rat skins in oitro using 60% (n=3),80% (n=5),andlOO% (n=4)ethanolinwateras donor phase solvent. Error bars indicate mean standard errors.

2.0

1 1.5

10

0.5

0.0

0 20 40 60 80

TIME(h)

Fig. 3. Cumulative release of LN through rat skins in vitro usingO% (n=3),20% (n=4),and40% (n=3) ethanolin water as donor phase solvent.

rat skin from pure water is quite low [O.Ol ,ug/ ( cm2 h ) 1. The flux increased when ethanol was added to 20%; from 40% to 100% ethanol, the steady-state flux was essentially unchanged [0.06 to 0.07 pg/ (cm2 h) 1. The cumulative re- lease plots for LN from the water/ethanol en- hancer systems are shown in Figs. 3 and 4. The steady-state flux of LN through rat skin in vitro using pure water, ethanol/water mixtures and pure ethanol is shown in Fig. 5. All the flux val- ues obtained using ethanol and ethanol/water mixtures were far below the target flux value of 0.4 ,ug/(cm* h) through rat skin. Therefore, a series of primary and secondary alkanols were

4

1 80% EtOH

0 20 40 60 80

TIME (h)

Fig. 4. Cumulative release of LN through rat skins in uitro using60% (n=3), 80% (n=5), and 100% (n=4) ethanol in water as donor phase solvent.

b 2

OOOI . , . , . I . 1 . I

0 20 40 60 80 100

L % ETHANOL IN WATER

Fig. 5. Steady-state flux of LN through rat skin in uitro. The donor phase enhancer composition consisted of 0 to 100% ethanol in water.

investigated for their ability to act as permea- tion enhancers for LN.

The steady-state flux of LN through rat skin in vitro using the neat n-alkanols ethanol, l- propanol, 1-butanol, 1-pentanol, 1-hexanol, and l-octanol as enhancers is given in Table 1. Also included in Table 1 is the steady-state flux of LN through rat skin in vitro from the secondary alkanols 2-propanol, 2-butanol, and 2-pen- tanol. The flux of LN through rat skin from l- butanol and 1-pentanol is shown in Fig. 6. The cumulative release of LN through the skins from 1-butanol and 1-pentanol is given in Fig. 7.

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TABLE 1

Steady-state flux of LN through rat skin in vitro from var- ious l-alkanols and 2-alkanols” and LN solubility in the alkanols at 32°C

Donor Steady-state flux phase [,&(cm’h)l

LN solubility

(mg/ml)h

ethanol 0.06 6.0 1 -propanol 0.36 7.0 1-butanol 0.70 4.7 1-pentanol 0.52 5.7 1 -hexanol 0.08 3.8 1-octanol 0.02 4.6 2-propanol 0.18 3.8 2-butanol 0.60 3.7 2-pentanol 0.26 4.3 propylene glycol 0.02 31.8

“n = 3 to 6 for each enhancer tested. “Measured at 32’ C.

0 20 40 60 60

TIME (h)

Fig. 6. Flux of LN through rat skins in oitro using pure l- butanol (n = 6) and neat 1-pentanol (n = 3 ) as enhancers. Error bars indicate mean standard errors.

There was an increase in flux from ethanol to 1-propanol to 1-butanol. The enhancement ef- fect diminished as the alkyl chain was in- creased beyond C,. The effect of alkyl chain length of the alkanol on steady-state flux is shown graphically in Fig. 8. Also included in Fig. 8 is the steady-state flux of LN through rat skin from the secondary alkanols. The flux pattern is the same except that the flux was lower for the secondary alkanols relative to the corre- sponding primary alkanols. Propylene glycol was also examined for its ability to improve the

40 7 1 -Butanol

30 -

20 -

10 -

0 0 20 40 60 60

TIME (hl

Fig. 7. Cumulative release of LN through rat skins in vitro using pure 1 -butanol (n = 6 ) and neat 1 -pentanol (n = 3 ) as enhancers.

2 2 z -I 0.60 -

& X 2 0.40 -

IL

2 Q ozo-

k7

L Q ! 0 12 3 4 5 6 7 6 9

ALKANOL CHAIN LENGTH

Fig. 8. Steady-state flux of LN through rat skins in vitro using enhancers of 1-alkanols and 2-alkanols.

flux of LN. The steady-state flux of LN through rat skin from neat propylene glycol is also shown in Table 1. There was very little flux enhance- ment with propylene glycol relative to water.

Permeability of LN through human cadaver skin

The permeability of LN through human ca- daver skin was also measured. The skin was ob- tained from the thigh. The enhancer used was pure ethanol. The flux of LN obtained (n = 3) from 40 to 50 h was 0.016 ,ug/ (cm’ h). How- ever, there was a considerable lag time in this experiment (30 to 40 h), therefore this flux

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value probably does not represent a steady-state value. Nonetheless, the flux obtained is reason- able considering the lower permeability of hu- man skin relative to rat skin [ 7,8].

Solubility of LN in alkanols

The solubility of LN in each of the solvents used as enhancers was measured at 32 ‘C. This was the temperature of the donor phase during the course of the transdermal flux experiments. The solubility data are presented in Table 1. The solubility of LN was very similar in all the al- kanols tested as enhancers except propylene glycol. The solubility of LN in pure water is very low (about 1.0 pug/ml at room temperature) and therefore difficult to measure accurately. There was no correlation between solubility and transdermal flux.

The octanol/water (0.05 M sodium phos- phate, pH 7.4) partition coefficient (K) of LN was measured as 5,000 (logK= 3.70).

DISCUSSION

Penetration enhancers and LN

The immediate goal of this research is to pre- pare a transdermal delivery system capable of delivering 25 ,ug of LN/day. To reach this goal, a penetration enhancer (or enhancers) is re- quired. An alternative would be to use prodrugs to modify the properties of the drug to increase permeability (see next paper in this issue). The experiments reported herein were designed to assess directly the effect of the enhancer on the barrier properties of rat skin towards LN. Sat- urated solutions of LN were used in all the ex- periments to maintain an equal (and maximum) driving force with all enhancer compositions [9]. Thus any differences in the skin permeability of LN from the various en- hancers are due to specific enhancer/skin in- teractions. The mechanism of action of the enhancers can be due to several factors: in-

creased partitioning of LN into the viable epi- dermis, enhanced partitioning into the stratum corneum, and increased diffusivity of LN due to the alkanols. Each of these possibilities is discussed below.

It is generally thought that percutaneous ab- sorption of most drugs is limited by the stratum corneum [lo]. This is true for most drugs. However, LN is an extremely hydrophobic compound: its water solubility is about 1 ppm at room temperature and the log of its octanol/ water partition coefficient is 3.70. It is there- fore conceivable that the hydrophilic viable ep- idermis is a significant barrier to the transdermal flux of LN. The mechanism of ac- tion of the alkanols can be explained to some degree by assuming that partitioning of LN into viable epidermis limits transdermal flux.

The alkanols studied are known to enter the skin [ 11-131. When they do so, they can alter the solubility of chemicals in the skin. If the rate-limiting step in LN absorption is the par- titioning of LN from the stratum corneum into the viable epidermis, then any chemical that can enter the viable epidermis, and increase the sol- ubility of LN in that phase, should increase the flux of LN through the skin. Because the solu- bility of LN in all the alkanols tested is nearly equivalent (see Table 3 ) , the amount of alkanol capable of entering the viable epidermis is probably important in contributing to the over- all flux enhancement of LN.

The water solubility of the alkanols studied as enhancers decreases as the alkyl chain length increases. The alkanols from ethanol to l-pen- tanol are miscible with water; 1-hexanol is only slightly soluble in water; and 1-octanol is essen- tially insoluble. Also, as the alkyl chain length increases there is an increase in the permeabil- ity of the n-alkanols through intact skin [ ll- 13 1. The barrier towards absorption of these al- kanols is apparently the stratum corneum [ 131. Once the alkanol has passed the stratum cor- neum, it must enter the viable epidermis, a pro- cess which is controlled primarily by the water solubility of that alkanol. Thus, the amount of

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alkanol entering the viable epidermis decreases accordingly as chain length increases [ 131.

The more alkanol that enters the skin (con- trolled by both the hydrophilic and lipophilic properties of the alkanol), the greater the permeability enhancement of LN. As the chain length increases beyond an optimum point (n- butanol in this case), the hydrophobic proper- ties of the alkanol limits its ability to partition into the viable epidermis. Hence the decreasing permeation enhancement with increasing chain length beyond n-butanol.

This proposed mechanism of permeation en- hancement does not preclude the possibility of other specific interactions of LN, the alkanols, and the various components of the skin. An- other possibility is that the alkanols are acting on the stratum corneum directly. The alkanols may be increasing the partitioning of LN into the stratum corneum, or they may be altering the diffusivity of the stratum corneum towards LN. The more hydrophobic alkanols may be ex- tracting lipids from the stratum corneum lead- ing to increased diffusion through this part of the skin. Additional experiments are required to determine where in the skin these enhancers are functioning and in what way they are alter- ing the permeability of the skin. Performing these experiments with skin without stratum corneum (stripped), and epidermis only, would help to separate the effects the alkanols have on the various strata of the skin.

Transdermal delivery of LN for fertility regulation

The target delivery rate of LN which will suppress estrus is 0.1 ,ug/(cm” h). The perme- ability of LN through human cadaver skin us- ing pure ethanol as an enhancer gave a flux about four times Iower than LN flux through rat skin with the same enhancer. Therefore, the target flux in the in vitro model employed is 0.4 pg/ ( cm2 h ). This value is used only to identify potential enhancer compositions for in uiuo testing. Of the enhancers tested in the present

study, l-propanol, l-butanol, 1-pentanol, and Z-butanol gave fluxes of LN greater than 0.4 fig/ (cm’ h) through rat skin. The best enhancer was 1-butanol. Its use as an enhancer in a transdermal delivery system for humans may be limited by contact dermatitis and its off odor. Of the remaining alkanols, 1-propanol appears most suited for use as an enhancer considering potential toxicity and irritation. As was seen with ethanol/water mixtures, it may be possi- ble to dilute 1-propanol or 1-butanol with ethanol while still retaining suf~~ient permea- bility enhancement. We have observed this with other enhancer combinations. Skin irritation studies in rabbits, as well as in uiuo permeabil- ity studies are currently underway.

ACKNOWLEDGEMENTS

This work was supported by the National In- stitute of Child Health and Human Develop- ment under Contract No. NOl-HD-5-2911. We would like to thank Ms. Anne Mac for her ex- cellent technical assistance during the course of this work.

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