Transdermal iontophoretic drug delivery: Mechanistic analysis and application to polypeptide delivery

Transdermal lontophoretic Drug Delivery: Mechanistic Analysis and Application to Polypeptide Delivery V. SRINIVASAN*, W. I. HIGUCHI*~~, S. M. SIMS*, A. H. GHANEM*,AND C. R. BEHL' Received September 6, 1988, from the 'Department of Pharmaceutics, 301 Skaggs Hall, University of Utah, Sait Lake Cip UT 841 72,and the Accepted for pub ication November 30, *Pharmaceutical Research & Development Department, Hoffmann-LaRoche Inc., Nutley, NJ 077 10. 1988.

~~

Three factors are of primary importance in determining the iontophoretic flux of a charged solute: the electrochemical potential gradient across the skin, an increase in skin Permeability to passive transport due to iontophoresis (loosely defined as skin damage),and a current-induced water flux. The latter two factors can also affect the transport of uncharged solutes during iontophoresis. A method of correcting for the skin damage effect is introduced. The contributions of the water transport effect relative to that of the applied voltage drop for charged solutes is estimated. It is shown that the water transport contribution is generally lower than the contribution due to the applied voltage drop. The observed iontophonetic flux of the enhancement factors due to the applied voltage drop alone are compared with the theoretical predictions based on the constant field assumption. It is shown that the theoretical predictions are higher than the experimental observations. This work also examines, for the first time, a synergism of iontophoresis and pretreatment with a chemical penetration enhancer as a means for delivering high molecular weight polypeptides. It is shown that a 2-h pretreatment with absolute ethanol followed by iontophoresis dramatically increases the permeability coefficient of insulin through human skin. Abstract

The transdermal delivery of many ionized drugs a t therapeutic levels is precluded by their slow rate of diffusion under a concentration gradient alone. Iontophoryis is the process of enhancing the rate of penetration of ions through skin by the application of a voltage drop across the skin. The magnitude of the flux enhancement depends primarily on the magnitude of the applied voltage drop; thus, it may be possible to elevate the flux of ionized drugs t o therapeutic levels by iontophoresis. Therefore, this noninvasive technique could serve as a substitute for chemical penetration enhancers. Alternatively, iontophoresis could synergize with a chemical penetration enhancer to potentially permit the transdermal delivery of high molecular weight polypeptides. A growing number of polypeptide drugs (e.g., insulin, growth hormone, etc.) either are, or will soon be, commercially available. Most of these polypeptides are susceptible to degradation by proteolytic enzymes and are thuv unsuitable for oral delivery. Parenteral administration is the preferred route, but because they are therapeutically effective only for a short duration, repeated injections are often required. Therefore, there is an urgent need for the development of nonparenteral routes of administration with controlledrelease characteristics. The ionization state of a polypeptide in solution depends on its isoelectric point (PI) and the pH of the solution. Many polypeptides at physiological pH exist as ions. Therefore iontophoresis may be one possible nonparenteral route for the administration of polypeptides. A synergism of iontophoresis and a chemical penetration enhancer may facilitate the transdermal delivery of high molecular weight polypeptides. Several published accounts of iontophoresis for local and systemic therapy are available.14 More Fecent efforts have 370 1 Journal of Pharmaceutical Sciences VoI. 78, No. 5, May 1989

been devoted to identifying the fundamental factors contributing to the iontophoretic flux and relating these factors to the transport properties of the membrane and the drug.513 Three factors appear to be of primary importance in determining the flux of an ionic drug iontophoresis: the electrochemical potential gradient across the skin, an increase in skin permeability due to iontophoresis (loosely defined as skin damage), and a current-induced water transport effect. The latter two factors can also affect the transport of uncharged solutes during iontophoresis.6 The increase in skin permeability to passive transport due to iontophoresis is well documented.- Srinivasan et a1.6 also present some experimental evidence to suggest that the increase in skin permeability of hairless mouse skin (HMS) occurs quite rapidly and is followed by a period during which the permeability of the skin can be considered to be relatively constant. This permits a n introduction of a correction factor that accounts for the skin damage effect and facilitates the estimation of the relative magnitudes of the effect of the applied voltage drop and the current-induced water flux. Using a polar uncharged solute (glucose),which should not be directly affected by the applied voltage drop, they have estimated the enhancement i n flux of glucose during iontophoresis attributable to the water flux. They have also shown that this effect is in the same direction as the current a t pH 7.4, and can either assist or oppose the flux of a charged solute. The effect of the electrochemical potential gradient on the flux of ions through a membrane has been modeled by several investigators.6J2J4 An equation predicting the enhancement in flux (relative to flux without the applied voltage drop) is given. This equation is derived on the assumption that skin damage and water transport effects are negligible and that the voltage drop across the membrane is linear. Experimental results have also been published5J3J" which suggest that the agreement with theory is reasonable at low applied voltage drops, but poor at higher voltage drops. These comparisons between theory and experiment were made without correcting the experimentally measured enhancement factors for the effects of skin damage and water transport. In this paper, a quantitative assessment is made of the relative contributions to the iontophoretic flux across hairless mouse skin (HMS) due to the applied voltage drop and the water flux for model charged solutes, using the four-electrode potentiostat system.5 The effect of skin damage is corrected for as described in Srinivasan et a1.,6 so that the contribution of the water flux relative to that of the applied voltage drop can be well estimated. Once the effect of the water flux and the skin damage have been corrected for, the actual enhancement in the flux (relative to passive diffusion) caused by the applied voltage drop alone can be estimated and compared with the theoretical prediction. This permits a more rigorous test of the validity of the theory. This work also examines, for the first time, a synergism of 0022-3549/89/0500-037050 l.OO/O 0 7989,American Pharmaceutical Association

iontophoresis and pretreatment with a known chemical penetrat:ion enhancer such1 as ethanol as a means for delivering a high molecular weight polypeptide. The effect of pretreatment of human skin (HS) with ethanol followed by iontophoresis on the permeability coefficient of insulin is reported.

Theoretical Section A lcommon starting point for the formal description of diffusion is the Nermt-P lanck equation, which for ideal soluti.onsand steady-state conditions is given by the following equai;ion:14 J. =

[

dCli -D.--

'dx

+--CiZiF d+] RTdx

where J, is the steady-state flux of species i, D, is the diffusivity of i, C , is the concentration of i, Z, is the charge on i, *is the electric potentia1,P is the Faraday constant, R is the gas constant, and T is the ahsolute temperature. The simplest and most frequently used assumption to facilitate the integration of eq 1is the constant field assumption of Goldman.16 With this assumption, eq 1 can be integrated to give an enhancement factor E (relative to the passive flux) which is given by eq 2:6J2,14

where PA+and Po are the iontophoretic and passive permeability coefficients, respectively, and K is defined as follows:

(3) where A$ is the voltage drop across the membrane. Implicit in eq 1 is the assumption that water flux and skin damage effects,are insignificant.

Experimental Section Materials-Tetraethylammo.nium bromide (TEAB) was selected as a model positively charged solute. It is a highly water soluble salt and dissociates in aqueous buffers into the monovalent TEA+ and Br-. Radiolabeled TEAB ([1-14C]TEAB,4.7 mCiimmo1) was obtained from New England Nuclear, Boston, MA, stored in ethanol, and air dried and reconstituted in the buffer before each experiment. Butyric acid and citric acid were chosen as model negatively charged solutes. Butyric acid, with pKo= 4.84, slhould be fully ionized to the butyrate anion at pH 7.4. Citric acid with three pK, values at pH 3.15, 4.78, and 6.4 can be expected to bte a trivalent anion at pH 8.0. Radiolabeled butyric acid ( [ l - 1 4 C l b ~ t y racid, i ~ 13.4 mCi/mmol) and [1,5-'4Clcitric acid (54.5 mCi/mmol) were also obtained from New England Nuclear. Butyric: acid was stored in ethanol and was air dried and reconstituted in the buffer before each experiment. Citric acid was stored in a n ethano'kwater solution (1:9) ant1 was used as received. Porcine insulin (methyl-14C, 0.024 mCiimg) was obtained from New England Nuclear, Unlabeled porcine insulin was obtained from Sigma Chemical, St. Louis, MO. Membranes-All experiments with TEAB, butyric acid, and citric acid were carried out with hairless mouse skin (HMS).Full-thickness HMS was excised from the abdomen of a 2-3-month-old male mouse according to the procedure of Durrheim et al.17 and was used immedi.ate1y for two simultaneous experiments. All t.he insulin experiments were carried out with human skin. B from the Department of Full-th.ickness human skin W ~ obtained Medicine, Division of Dermatology, University of Utah. The skin, obtained from tummy-tuck surgely, was dermatomed to 500-/.an thickness and stored in a tissue culture medium at 4 "C. The details of the pretreatment undergone by the skin, from s u r s cal excision to

dermatome and storage, is described elsewhere.5 The human skin used in these experiments was 4-10 d old. Buffers/Electrolytes-All the TEAB experiments were conducted in pH 7.4 isotonic phosphate buffered saline (PBS). The citric acid experiments were conducted in pH 8.0 isotonic PBS. The butyric acid experiments were conducted in PBS, pH 7.4, with a n ionic strength of 0.1 M. The insulin experiments were all conducted in pH 8.0 isotonic PBS. Insulin has a pI of -5.3 and can be expected to be negatively charged a t this pH. All buffers were prepared in distilled, deionized water according to standard procedures.18 All the experiments with radiolabeled compounds were conducted with trace amounts of the permeant on the donor side, so that the concentration and ionic strength of the donor and receiver solutions were practically identical. All experiments were carried out at 37 "c. Four-Electrode Potentiostat System-This system is ideally suited to maintain a constant voltage drop across a membrane in a two-chamber diffusion cell and was developed by Masada et al.15 Figure 1 is a schematic of the four-electrode potentiostat and its associated two-chamber diffusion cell. The potentiostat maintains the voltage drop across the two reference electrodes (placed very close to the membrane on either side) at the set value by driving the required current through the membrane with the help of the counter electrodes (Pt wires). Figure 2 shows one half-cell of the two-chamber diffusion cell with its associated reference and counter electrode. The fourelectrode potentiostat system is described in detail by Srinivasan et al.,5 who also compare it with the conventional two-electrode, constant current system. Experimental Procedures for Model Solutes-The experimental procedure in this work for the experiments with TEAB, citric acid, and butyric acid is identical to the three-stage permeation experiment described in detail in Srinivasan e t a1.6 In stage I, the initial passive permeability coefficient, Po, for the permeant through HMS was determined. In stage 11, a voltage drop, A+, was applied across the skin and the iontophoretic permeability coefficient, PA, of the permeant through the same skin was determined. For the TEAB experiments, the anode was in the donor side and the cathode in the receiver side. For the butyric and citric acid experiments, the polarity was reversed. Stage I1 lasted for 1 h. The applied voltage drop was then turned off and a passive permeability coefficient after iontophoresis,P&was determined (Stage 111).For each stage, the permeability coefficient was calculated from the following equation:

p = - - 1 dQ CDA dt

(4)

where P is the permeability coefficient (cmis), CD is the donor side concentration (dpmicm3),A is the area for diffusion (cm'), and (dQldt) is the slope of the permeation profile after correcting for sampling effects (dpmis; Q is the cumulative sampling corrected radioactivity on the receiver side as function of time t). The sampling, assay, and data analysis procedures have been described in detail elsewhere.5.6

CEI

RE1

RE2

E 2

FOUR-ELECTROOE

POTENTIOSTAT

CE = COUNTER ELECTROOE R E = REFERENCE EFECTROOE

MEMBRANE

Figure 1-Schematic of the diffusion cell and the four-electrode system for iontophoresis. Journal of Pharmaceutical Sciences / 371 Vol. 78, No. 5, May 1989

L

0.50V 0.250V 0.125V

REFERENCE ELECTRODE

+

E

4001 300

m

1

""I

IER PORT

INTER

100

+

+ + +

0

1

0

'

1

10

0

0

C I o n

'

+

+

1

'

30

20

0

1

'

1

50

40

'

1

60

TIME (MINUTES)

JACKET-'

Figure 2-Schematic

of a diffusion half-cell and its associated reference and counter electrodes.

Figure W e l l current versus time at constant applied voltage drop for citrate ion through HMS.

Experimental Procedure for Insulin-The effect of ethanol pretreatment followed by iontophoresis on the permeability coefficient of insulin (MW = 5766) through human skin was investigated. The pretreatment procedure consisted of assembling the diffusion cells with the human skin in the usual manner,5.6 filling both chambers with absolute ethanol, and maintaining the cells a t 37°C with stirring for 2 h. The ethanol was then drained, the two chambers rinsed thoroughly with the buffer, and the transport experiment carried out to determine Po, PO,,,v and Po 5v in sequence. Control experiments were carried out without ethanol pretreatment under identical conditions.

rapid increase in current initially (within -5-10 min), followed by a relatively steady current afterwards. The samples for the PA, calculation were all withdrawn during the last 30 minutes of stage 11. In all the experiments, there was a definite increase in the permeability coefficient from stage I to stage 111, suggesting iontophoresis had increased the permeability of the skin to passive transport. This is consistent with the observations of other investigators.- It was first thought that the increase in current may correlate with the increase in skin permeability; if this were true, Pb would clearly be the appropriate passive permeability coefficient for the calculation of the enhancement factor E in eq 2. Experiments are underway to examine this possible correlation between the increase in current and the increase in permeability. Preliminary results suggest that the increase in permeability may be more gradual than the increase in the mrrent. Even in this case, it is believed that P; would be more appropriate than Po for calculating the enhancement factor since P,, is determined during the last 30 minutes of stage 11. Permeability Experiments with Tetraethylammonium Bromide (TEAB) and Hairless Mouse Skin (HMSl-Figure 5 shows a typical permeation profile for a three-stage experiment. Stage I is for Po determination, stage I1 is for P A , determination at 0.5 V, and stage I11 is for P A determination.

ResuIts Current-Voltage Relationship for Hairless Mouse Skin ( H M S t T h e four-electrode potentiostat system maintains a constant voltage drop across a membrane and continuously monitors and displays the current flowing through the membrane.5 Figure 3 is a plot of the cell current as a function of time for three different applied voltage drops across HMS during the iontophoresis ofTEAB (data obtained during stage I1 of three different experiments). Figure 4 is a similar plot for three different applied voltage drops during the iontophoresis of citric acid. The profiles are all qualitatively similar-a 0.50V 0.250V 0.125V

+

s n Y

H

50000

40000

v)

I-

3

30000

0 K

W

50

o

0

-I

J UI

+

u

1

0

10

20

~

+ 1

30

~

40

'

50

1

60

Figure 3-Cell current versus time at constant applied voltage drop for TEA ion through HMS.

-1

1

;

I

I

I

I

I

1.I STAGE I

I

I

I

= I

.

I

{ I = I

10000

STAGE 111

I

I I

W

+ 1

TIME (MINUTES)

372 / Journal of Pharmaceutical Sciences Vol. 78,No. 5, May 1989

20000

I

STAGE II

'

U 1

0

8

10

12

14

16

TIME (HOURS) Figure &Typical permeation profile for a three-stage experiment (0.0 V-0.5 V 4 . 0 V) for TEA ion through HMS.

Table U-Permeablllty

Coefficient of Tetraethylammonium (TEA) Ion through Hairless Mouse Skin (HMS)e

-

Applied Voltage

No. of Experiments

(

4

9

v

0.125 0.250 0.500

:I

2 3 a

4

Permeability Coefficient, cm/sb Po

P

p6,

(2.9? 0.9)x lo-: (1.1 0.2)x 10(6.4+- 4.1)X lo-'

(1.a2 0.5)x 10-7 (1.95 0.4)x lo-' (3.6t 2.5)X lo-'

(5.32 1.7)x (2.8f 0.3)X (2.02 1.7)x 10-7

*

The electrolyte was pH 7.4i:;otonic PBS at 37 "C. Mean

* SD.

The best-fit straight line for each stage showed a very good correlation coefficient (>0.98) for all the experiments. Table I lists the Po,P,,,,, and P 6 values for TEA ion through HMS for three different applied voltage drops. To better interpret the results, the following quantities can be defined6

(5)

= (P**lPo) E , = (PA*IPb) DF = (ElIE21= (PyPo)

(6)

(7)

where E , is the total enhancement factor over passive diffusion alone, E, is an enhancement factor after introducing a correction for the skin permeability increase due to iontophoresis, and DF is a damage factor which measures the extent, of increase in the skin permeability. Table I1 lists E l , E,, and DF values for TEAB. The values of DF are always >1,indicating that the skin becomes more permeable due to the applied voltage drop. Figure 6 is a plot of E l and E , as a function of the applied voltage drop across HMS. The solid line is the theoretical prediction of eq 2: El deviates increasingly with increase in the voltage drop, while E , is in good agreement with the theory. Also shown in Figure 6, for comparison, are E2values for an uncharged polar solute such as glucose under the same experimental conditions as TEAB. The E , values far gliicose are taken from Srinivasan et a1.6 The E, values for glucose are much less than the corresponding values for TElA13under identical conditions. Table Il-Enhancement Factor and Damage Factor for Tetraethylammonium (TEA) lor1 through Hairless Mouse Skin (HW

Applied Voltage

Enhancement Factora Eq 2

(A$),

0.125 0.250 0.500

4.7 9.4 18.7

Damage Factor (DF)a

€2

€1

*

6.2 0.3 17.5 f 6.7 55.8 t 6.8

3.3 ? 0.2 6.8t 0.7 19.52 3.1

1.92 0.1 2.5 2 0.7 2.92 0.6

aMean f SD.

P z W 0

0

r z w

-

o 0.00

0.25

,

P 0.50

EI TEA+ EZTEA+ E2GLUCOSE

I

0.75

APPLIED VOLTAGE DlROP (VOLTS)

Figure &Enhancement factor as a function of the applied voltage drop for TEA ion and glucose throiugh HMS. The solid line is the prediction of eq 2.

Permeability Experiments with Citric Acid and Hairless Mouse Skin (HMSbTable I11 lists the Po,PA,,,,and P A values for citrate ion through HMS for three different applied voltage drops. The quantities E l , E,, and DF are listed in Table IV. Figure 7 is a plot of E, and E , as a function of the applied voltage drop. The solid line is the prediction of eq 2 assuming Z = -3. The El values deviate increasingly from theory with increasing voltage drop, while E, is lower than the theoretical prediction by about a factor of two to three. Permeability Experiments with Butyric Acid and Hairless Mouse Skin (HMSbFigure 8 is a plot of E , and E , as a function of the applied voltage drop. As with citric acid, the E , values are lower than the theoretical predictions by about a factor of two to three. Permeability Experiments with Insulin and Human Skin-Control experiments without ethanol pretreatment of the human skin showed no measurable flux of insulin at applied voltage drops of 0.25 and 0.5 V. Therefore, a combination of ethanol pretreatment followed by iontophoresis was undertaken. Figure 9 shows the effect of ethanol pretreatment on the passive permeability coefficient (Po)of a polar solute like TEAB. Pretreatment with 100%ethanol for 2 h appears to enhance the passive permeability coefficient of TEAB by -100-fold. Table V summarizes the permeability coefficients and enhancement factors for insulin through ethanol retreated human skin. A measurable Po value of 3.5 x 10- (P c d s was obtained after ethanol pretreatment. The application of 0.25 and 0.5 V across the skin resulted in significant enhancements in the permeability coefficients of insulin. Stability of the Permeating Species in the Insulin Experiments--In the transport experiments with radiolabeled insulin, the donor concentration of insulin was very low,